Antibodies directed to αVβ6 and uses thereof

ABSTRACT

Targeted binding agents, such antibodies directed to the antigen αVβ6 and uses of such agents are described. In particular, fully human monoclonal antibodies directed to the antigen αVβ6 are disclosed. Nucleotide sequences encoding, and amino acid sequences comprising, heavy and light chain immunoglobulin molecules, particularly sequences corresponding to contiguous heavy and light chain sequences spanning the framework regions and/or complementarity determining regions (CDR&#39;s), specifically from FR1 through FR4 or CDR1 through CDR3 are disclosed. Hybridomas or other cell lines expressing such immunoglobulin molecules and monoclonal antibodies are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 12/782,335filed on May 18, 2010, (now U.S. Pat. No. 8,398,975 issued on Mar. 19,2013), U.S. application Ser. No. 12/782,335 is a continuation of U.S.application Ser. No. 11/833,486 filed on Aug. 3, 2007. U.S. applicationSer. No. 11/833,486 claims benefit under 35 U.S.C. §119(e) of thefollowing U.S. Provisional Application No. 60/835,559 filed Aug. 3,2006. Each of the above listed applications is incorporated by referenceherein in its entirety for all purposes.

REFERENCE TO THE SEQUENCE LISTING

This application incorporates by reference a Sequence Listing submittedwith this application as text file entitled A5B6100US3_seq_listingcreated on Jan. 15, 2013 and having a size of 83 kilobytes.

FIELD

The invention relates to monoclonal antibodies against alphaVbeta6integrin (αVβ6) and uses of such antibodies. In some embodiments, theinvention relates to fully human monoclonal antibodies directed to αVβ6.The described antibodies are useful as diagnostics and for the treatmentof diseases associated with the activity and/or overproduction of αVβ6.

BACKGROUND

The integrin superfamily includes at least 24 family members consistingof heterodimers that utilize 18 alpha and 8 beta chains (Hynes, (2002)Cell 110: 673-87). This family of receptors is expressed on the cellsurface and mediates cell-cell and cell-extracellular matrixinteractions that regulate cell survival, proliferation, migration, anddifferentiation as well as tumor invasion and metastasis(ffrench-Constant and Colognato, (2004) Trends Cell Biol. 14: 678-86).Integrins bind to other cellular receptors, growth factors andextracellular matrix proteins, with many family members havingoverlapping binding specificity for particular proteins. This redundancymay ensure that important functions continue in the absence of aparticular integrin (Koivisto et al., (2000) Exp. Cell Res. 255: 10-17).However, temporal and spacial restriction of expression of individualintegrins with similar specificity has also been reported and may alterthe cellular response to ligand binding (Yokosaki et al., (1996) J.Biol. Chem. 271: 24144-50; Kemperman et al., (1997) Exp. Cell Res. 234:156-64; Thomas et al., (2006) J. Oral Pathol. Med. 35: 1-10).

The integrin family can be divided into several sub-families based onligand specificity of the heterodimers. One subfamily consists of all ofthe integrins that recognize and bind the RGD tripeptide. Thesereceptors include the αIIb/β3 and all of the αV heterodimers (Thomas etal., (2006) J. Oral Pathol. Med. 35: 1-10). While the αV chain can pairwith 5 known beta chains, several of these beta chains can only pairwith αV. The β6 chain is selective for heterodimerization to αV and thispair binds extracellular matrix and cytokine proteins with either highor low affinity. αVβ6 binds to the RGD motifs on both TGFβ1LAP andTGFβ3LAP latent complexes and activates them (Munger et al., (1999) Cell96: 319-328; Annes et al., (2002) FEBS Letters 511: 65-68). However, itdoes not bind to or activate TGFβ2LAP, which does not have thetri-peptide (Ludbrook et al., (2003) Biochem. J. 369: 311-18).αVβ6-mediated activation of TGFβ requires the latent TGFβ bindingprotein 1 (LTBP1), which tethers the latent TGFβ complex to theextracellular matrix. Activation is proposed to result from aconformation change induced as the TGFβLAP is held between the cell andthe matrix by αVβ6 and LTBP1, respectively (Keski-Oja et al., (2004)Trends Cell Biol. 14: 657-659; Annes et al., (2004) J. Cell Biol. 165:723-34). The picoMolar binding affinity of αVβ6 for the TGFβLAPcomplexes is the highest for any of its known ligands. Other ligands forαVβ6 include fibronectin, tenascin, vitronectin and osteopontin (Busk etal., (1992) J. Biol. Chem. 267: 5790-6; Prieto et al., (1993) PNAS 90:10154-8; Huang et al., (1998) J. Cell. Sci. 111(Pt 15): 2189-95;Yokosaki et al., (2005) Matrix Biol. 24: 418-27). The binding affinityof αVβ6 for these extracellular matrix proteins is low affinity and inthe nanoMolar range.

Expression of αVβ6 integrin is restricted to areas of active tissueremodeling in the adult, specifically on the epithelia of healing woundsand at the edge of invading tumors (Breuss et al., (1995) J. Cell Sci.108: 2241-51). Keratinocytes at the wound edge upregulate the expressionof αVβ6 during their migration into the wound, but expression remainshigh after the edges of the wound epithelium have joined (Breuss et al.,(1995) J. Cell Sci. 108: 2241-51; Haapasalmi et al., (1996) J. Invest.Dermatol. 106: 42-48). The wound extracellular matrix containsfibronectin, tenascin and vitronectin, all of which are ligands for αVβ6(Busk et al., (1992) J. Biol. Chem. 267: 5790-6; Koivisto et al., (1999)Cell Adhes. Commun. 7: 245-57; Hakkinen et al., (2000) J. Histochem.Cytochem. 48: 985-98). In addition, αVβ6 upregulates the expression ofthe matrix metalloproteinase, MMP-9, that can degrade Type IV collagenand promote cell movement (Niu et al., (1998) Biochem. Biophys. Res.Com. 249: 287-91; Agrez et al., (1999) Int. J. Can. 81: 90-97; Thomas etal., (2001) Int. J. Cancer 92: 641-50; Gu et al., (2002) Br. J. Can. 87:348-51). Based on its expression pattern in wounds and in vitro studies,αVβ6 may have dual roles to promote keratinocyte migration during woundclosure and later to resolve the wound through the activation of TGFβ.The activation of TGFβ by αVβ6 would contribute to wound resolutionthrough the regulation of re-epithelialization, suppression ofinflammation and promotion of connective tissue regeneration and scarformation (Thomas et al., (2006) J. Oral Pathol. Med. 35: 1-10). In vivowound studies using beta 6 null mice indicated that wounds healed butthere was a markedly increased inflammatory response in the skin. Woundclosure and keratinocyte activity was likely unaffected by the loss ofαVβ6 because of the expression of other integrin family members (Huanget al., (1996) J. Cell Biol. 133: 921-8). The inflammatory infiltrate inthe beta 6 null mouse wounds resembled those from TGFβ1 null mice,suggesting that there was insufficient activity of this cytokine tosuppress the immune response in the absence of αVβ6 (Shull et al.,(1992) Nature 359: 693-9; Thomas et al., (2006) J. Oral Pathol. Med. 35:1-10).

Analysis of the beta 6 null mice in lung injury and kidney diseasemodels has also identified a role for αVβ6 in fibrosis. Lung fibrosis inthe beta 6 null mice was inhibited in a bleomycin injury model (Mungeret al., (1999) Cell 96: 319-328). These animals also were protected froman MMP12 dependent emphasema-like phenotype (Morris et al., (2003)Nature 422: 169-73). Both disease phenotypes are dependent on theactivation of TGFβ (Munger et al., (1999) Cell 96: 319-328). Inhibitionof αVβ6 integrin-mediated TGFβ activation was also hypothesized topromote pulmonary edema in the early phase response to acute lung injury(Pittet et al., (2001) J. Clin. Invest. 107: 1537-44). Beta 6 null micewere also protected from fibrosis in a kidney disease model, where TGFβactivation is essential for the development of tubulointerstitialfibrotic lesions (Ma et al., (2003) Am. J. Pathol. 63: 1261-73).

In addition to its expression in wound healing, the αVβ6 integrin isupregulated at the periphery of many human tumors. αVβ6 expression hasbeen reported in oral (Breuss et al., (1995) J. Cell Sci. 108: 2241-51;Jones et al., (1997) J. Oral Pathol. Med. 26: 63-8; Hamidi et al.,(2000) Br. J Cancer 82: 1433-40; Regezi et al., (2002) Oral Oncology 38:332-6; Impola et al., (2004) J. Pathol. 202: 14-22) and skin squamouscell carcinomas, as well as carcinomas of the lung (Smythe et al.,(1995) Can. Met. Rev. 14: 229-39), breast (Arhiro et al., (2000) BreastCan. 7: 19-26), pancreas (Sipos, et al., (2004) Histopathology 45:226-36), stomach (Kawashima et al., (2003) Pathol. Res. Pract. 199:57-64), colon (Bates et al., (2005) J. Clin. Invest. 115: 339-47), ovary(Ahmed et al., (2002) Carcinogenesis 23: 237-44; Ahmed et al., (2002) J.Histochem. Cytochem. 50: 1371-79), and salivary gland (Westernoff etal., (2005) Oral Oncology 41: 170-74). In many of these reports theexpression of αVβ6 correlated with increasing tumor grade (Ahmed et al.,(2002) J. Histochem. Cytochem. 50: 1371-79; Arihiro et al., (2000)Breast Can. 7: 19-26), eventual metastases to lymph nodes (Kawashima etal., (2003) Pathol. Res. Pract. 199: 57-64; Bates et al., (2005) J.Clin. Invest. 115: 339-47), or poor prognosis (Bates et al., (2005) J.Clin. Invest. 115: 339-47). The most well studied tumor type is oralsquamous cell carcinoma, where investigators have also examined αVβ6 inpre-cancerous lesions and correlated its expression with progression tomalignancy (Hamidi et al., (2000) Br. J Cancer 82: 1433-40). The linkbetween αVβ6 expression and tumor progression has also been investigatedin colon carcinoma where the presence of integrin correlated with theepithelial-to-mesenchymal transition (EMT) of colon cells in an in vitromodel (Brunton et al., (2001) Neoplasia 3: 215-26; Bates et al., (2005)J. Clin. Invest. 115: 339-47). The EMT is a normal developmental processthat enables epithelial cells to leave their home tissue and migrate outto new areas (Thiery and Sleeman, (2006) Nat. Rev. Mol. Cell Biol. 7:131-42). It is marked by an increase in the expression of proteins thatpromote the migration and invasion of cells, such as matrix proteases,cytokines like TGFβ and a variety of cellular adhesion molecules,including integrins (Zavadil and Bottinger, (2005) Oncogene 24:5764-5774). The expression of EMT markers has also been identified intumors, particularly in aggressively invasive and metastatic carcinomas.The ability of αVβ6 to promote adhesion to interstitial fibronectin,upregulate the expression of MMP-9 and other matrix proteases and toactivate TGFβ indicates it may facilitate the EMT of malignant cells andtumor progression (Bates and Mercurio, (2005) Cancer Bio. & Ther. 4:365-70).

Animal and in vitro models of human cancer have implicated αVβ6 mediatedsignal transduction in the promotion of cell proliferation andinhibition of apoptosis. The residues within the C-terminus of the beta6 chain that promote proliferation of αVβ6-transfected SW480 colon tumorcells in a collagen gel matrix in vitro were identified. Compared to thefull-length β6 transfected SW480 cells, the β6 deletion mutant hadmarkedly reduced ability to grow sub-cutaneously in Nude mice (Agrez etal., (1994) J. Cell. Biol. 127: 547-56). In an oral cancer cell linethat stably expressed αVβ6, binding to fibronectin resulted in therecruitment and activation of the Fyn kinase by the beta 6 subunit.Downstream signal transduction resulted in the production of MMP-3,promoted cell proliferation in vitro, tumor invasion in an orthotopicmodel, and metastasis in a tail vein injection model (Li et al., (2003)J. Biol. Chem. 278: 41646-53).

Suppression of apoptosis, like cell proliferation, is another way thatαVβ6 may promote tumor growth. Normal stratified squamous epitheliaexpress the αVβ5 integrin but down-regulate it and upregulate αVβ6expression upon transformation to carcinomas. Using carcinoma cell linesthat over-expressed αVβ5, αVβ6 expression was shown to preventsuspension-induced cell death (anoikis) in vitro (Janes and Watt, (2004)J. Cell Biol. 166: 419-31). Apoptosis inhibition has also been observedin vitro in ovarian cancer cell lines treated with cisplatin, which mayrepresent a mechanism for drug resistance of these tumors in vivo (Wu etal., (2004) Zhonghua Fu Chan Ke Za Zhi 39: 112-14).

A number of investigators have developed therapeutics to target αVβ6activity in fibrosis and cancer. A murine antibody with specificity forthe αVβ6, αVβ3 and αVβ5 integrins was shown to prevent adhesion of HT29colon carcinoma cells to vitronectin and fibronectin in vitro (Lehmannet al., (1994) Can. Res. 54: 2102-07). Another murine antibodytherapeutic specific for the human αVβ6 protein was demonstrated toinhibit the invasive growth of HSC-3 oral carcinoma cells in a transoralxenograft tumor model in mice (Xue et al., (2001) Biochem. Biophys. Res.Com. 288: 610-18). A series of human αVβ6 specific antibodies wereraised using the beta 6 null mouse model as the host. These antibodieswere able to block both TGFβLAP and fibronectin binding to integrin invitro (Weinreb et al., (2004) J. Biol. Chem. 279: 17875-87). They alsodemonstrated significant tumor growth inhibition in a human pharyngealcancer xenograft model (Leone et al., (2003) Proc. of the Am. Assoc.Can. Res. 44, Abstract #4069).

In addition to function blocking antibodies the creation of apeptidomimetic inhibitor of the human αVβ6 integrin has been reported.This compound was shown to inhibit UCLAP-3 cell binding to fibronectinwith an IC50 in the 200 nM range with additional activity to block αVβ5and αVβ3 integrin-mediated cell binding to vitronectin in the 3-20 uMrange, respectively (Goodman et al., (2002) J. Med. Chem. 45: 1045-51).

Another recently described role for αVβ6 is as a cellular receptor forviral pathogens. It mediates the binding of the viral capsid forfoot-and-mouth disease virus and the Coxsackievirus 9 to enable viralentry in vitro (Miller et al., (2001) J. Virol. 75: 4158-64; Williams etal., (2004) J. Virol. 78: 6967-73). Both foot-and-mouth disease virusand Coxsackievirus 9 capsid proteins contain an RGD sequence that isrecognized by multiple integrin family members. Viral entry of bothpathogens is blocked by antibody to αVβ6 integrin (Williams et al.,(2004) J. Virol. 78: 6967-73).

SUMMARY

The invention is generally directed to targeted binding agents that bindto αVβ6. Embodiments of the invention relate to fully human targetedbinding agents that specifically bind to αVβ6 and thereby inhibitbinding of ligands to αVβ6. The targeted binding agents also inhibittumor cell adhesion. In addition, the targeted binding agents are usefulfor reducing tumor growth. Mechanisms by which this can be achieved caninclude and are not limited to either inhibiting binding of a ligand toits receptor αVβ6, abrogation of intereactions with ligands such asTGFβLAP, thereby reducing the effective concentration of αVβ6.

In one embodiment of the invention, the targeted binding agent is afully human antibody that binds to αVβ6 and prevents αVβ6 binding toligands of αVβ6. Examples of ligands of αVβ6 include TGFβLAP,fibronectin, tenascin, vitronectin and osteopontin. The antibody maybind αVβ6 with a K_(d) of less than 35 nM, 25 nM, 10 nM, or 60 pM.

Yet another embodiment is a fully human antibody that binds to αVβ6 andinhibits greater than 80%, 85%, 90% or 99% of TGFβ-LAP mediated adhesionof HT29 cells at antibody concentrations as low as 1 μg/ml or less.

Yet another embodiment is a fully human antibody that binds to αVβ6 andinhibits TGFβ3-LAP mediated adhesion of HT29 cells with an IC₅₀ of lessthan 0.070 μg/ml.

The targeted binding agent (i.e. an antibody) may comprise a heavy chainamino acid sequence having a complementarity determining region (CDR)with one of the sequences shown in Table 8 or Table 29. In oneembodiment the targeted binding agent may comprise a sequence comprisingany one of a CDR1, CDR2 or CDR3 sequence as shown in Table 8 or Table29. In another embodiment the targeted binding agent may comprise asequence comprising any two of a CDR1, CDR2 or CDR3 sequence as shown inTable 8 or Table 29 (that is either a CDR1 and CDR2, a CDR1 and CDR3 ora CDR2 and CDR3). In another embodiment the targeted binding agent maycomprise a sequence comprising a CDR1, CDR2 and CDR3 sequence as shownin Table 8 or Table 29. It is noted that those of ordinary skill in theart can readily accomplish CDR determinations. See for example, Kabat etal., Sequences of Proteins of Immunological Interest, Fifth Edition, NIHPublication 91-3242, Bethesda Md. (1991), vols. 1-3.

In another embodiment the targeted binding agent (i.e. an antibody) maycomprise a light chain amino acid sequence having a complementaritydetermining region (CDR), CDR1, CDR2 or CDR3 sequences as shown in Table9 or Table 30. In another embodiment the targeted binding agent maycomprise a sequence comprising any two of a CDR1, CDR2 or CDR3 sequenceas shown in Table 9 or Table 30 (that is either a CDR1 and CDR2, a CDR1and CDR3, or a CDR2 and CDR3). In another embodiment the targetedbinding agent may comprise a sequence comprising a CDR1, CDR2 and CDR3sequence as shown in Table 9 or Table 30.

The targeted binding agent of the invention may comprise anantigen-binding site within a non-antibody molecule, normally providedby one or more CDRs e.g. a set of CDRs in a non-antibody proteinscaffold, as discussed further below.

In another embodiment the targeted binding agent comprises a sequencecomprising any one of a CDR1, CDR2 or CDR3 sequence of fully humanmonoclonal antibodies sc 264 RAD, sc 264 RAD/ADY, sc 188 SDM, sc 133, sc133 TMT, sc 133 WDS, sc 133 TMT/WDS, sc 188, sc 254, sc 264 or sc 298.

In another embodiment the targeted binding agent comprises any two of aCDR1, CDR2 or CDR3 sequence of fully human monoclonal antibodies sc 264RAD, sc 264 RAD/ADY, sc 188 SDM, sc 133, sc 133 TMT, sc 133 WDS, sc 133TMT/WDS, sc 188, sc 254, sc 264 or sc 298 (that is either a CDR1 andCDR2, a CDR1 and CDR3 or a CDR2 and CDR3).

In another embodiment the targeted binding agent comprises a CDR1, CDR2and CDR3 sequence of fully human monoclonal antibodies sc 264 RAD, sc264 RAD/ADY, sc 188 SDM, sc 133, sc 133 TMT, sc 133 WDS, sc 133 TMT/WDS,sc 188, sc 254, sc 264 or sc 298.

In one embodiment of the invention, the targeted binding agent is anantibody. In one embodiment of the invention, the targeted binding agentis a monoclonal antibody. In one embodiment of the invention, thetargeted binding agent is a fully human monoclonal antibody.

Another embodiment of the invention comprises an antibody that binds toαVβ6 and comprises a light chain amino acid sequence comprising any oneof a CDR1, CDR2 or CDR3 sequence as shown in Table 9 or Table 30.Another embodiment of the invention comprises an antibody that binds toαVβ6 and comprises a light chain amino acid sequence comprising any twoof a CDR1, CDR2 or CDR3 sequence as shown in Table 9 or Table 30 (thatis either a CDR1 and CDR2, a CDR1 and CDR3 or a CDR2 and CDR3). Anotherembodiment of the invention comprises an antibody that binds to αVβ6 andcomprises a light chain amino acid sequence comprising a CDR1, a CDR2and a CDR3 sequence as shown in Table 9 or Table 30. In certainembodiments the antibody is a fully human monoclonal antibody.

Yet another embodiment of the invention comprises an antibody that bindsto αVβ6 and comprises a heavy chain amino acid sequence comprising anyone of a CDR1, CDR2 or CDR3 sequence as shown in Table 8 or Table 29.Another embodiment of the invention comprises an antibody that binds toαVβ6 and comprises a heavy chain amino acid sequence comprising any twoof a CDR1, CDR2 or CDR3 sequence as shown in Table 8 or Table 29 (thatis either a CDR1 and CDR2, a CDR1 and CDR3 or a CDR2 and CDR3). Anotherembodiment of the invention comprises an antibody that binds to αVβ6 andcomprises a heavy chain amino acid sequence comprising a CDR1, a CDR2and a CDR3 sequence as shown in Table 8 or Table 29. In certainembodiments the antibody is a fully human monoclonal antibody.

One embodiment of the invention comprises one or more of fully humanmonoclonal antibodies sc 264 RAD, sc 264 RAD/ADY, sc 188 SDM, sc 133, sc133 TMT, sc 133 WDS, sc 133 TMT/WDS, sc 188, sc 254, sc 264 or sc 298which specifically bind to αVβ6, as discussed in more detail below.

Yet another embodiment is an antibody that binds to αVβ6 and comprises alight chain amino acid sequence having a CDR comprising one of thesequences shown in Table 9 or Table 30. Another embodiment is anantibody that binds to αVβ6 and comprises a heavy chain amino acidsequence having a CDR comprising one of the sequences shown in Table 8or Table 29. In certain embodiments the antibody is a fully humanmonoclonal antibody.

A further embodiment is an antibody that binds to αVβ6 and comprises aheavy chain amino acid sequence having one of the CDR sequences shown inTable 8 or Table 29 and a light chain amino acid sequence having one ofthe CDR sequences shown in Table 9 or Table 30. In certain embodimentsthe antibody is a fully human monoclonal antibody.

A further embodiment of the invention is a targeted binding agent (i.e.an antibody) that competes for binding to αVβ6 with the antibodies ofthe invention. In one embodiment, said targeted binding agent comprisesa heavy chain amino acid sequence having at least one of the CDRsequences shown in Table 8 or Table 29 and a light chain amino acidsequence having at least one of the CDR sequences shown in Table 9 orTable 30.

A further embodiment of the invention is a targeted binding agent thatbinds to the same epitope on αVβ6 as the antibodies of the invention. Inone embodiment, said targeted binding agent comprised a heavy chainamino acid sequence having at least one of the CDR sequences shown inTable 8 or Table 29 and a light chain amino acid sequence having atleast one of the CDR sequences shown in Table 9 or Table 30.

In another embodiment the targeted binding agent comprises a sequencecomprising any one of a CDR1, CDR2 or CDR3 sequence as shown in Table 8or Table 29 and any one of a CDR1, CDR2 or CDR3 sequence as shown inTable 9 or Table 30. In another embodiment the targeted binding agentcomprises any two of a CDR1, CDR2 or CDR3 sequence shown in Table 8 orTable 29 and any two of a CDR1, CDR2 or CDR3 sequence as shown in Table9 or Table 30 (that is either a CDR1 and CDR2, a CDR1 and CDR3 or a CDR2and CDR3). In another embodiment the targeted binding agent comprises aCDR1, CDR2 and CDR3 sequence as shown in Table 8 or Table 29 and a CDR1,CDR2 and CDR3 sequence as shown in Table 9 or Table 30.

In some embodiments, a binding agent of the invention may comprise anantigen-binding site within a non-antibody molecule, normally providedby one or more CDRs e.g. a set of CDRs in a non-antibody proteinscaffold, as discussed further below.

A still further embodiment is an antibody that binds to αVβ6 andcomprises an amino acid sequence having one or more corrective mutationswhere the antibody sequence is mutated back to its respective germlinesequence. For example, the antibody can have a sequence as shown in anyof Tables 10-13.

The invention further provides methods for assaying the level of αVβ6 ina patient or patient sample, comprising contacting an anti-αVβ6 antibodywith a biological sample from a patient, and detecting the level ofbinding between said antibody and αVβ6 in said sample. In more specificembodiments, the biological sample is blood, or plasma.

In other embodiments the invention provides compositions comprisingtargeted binding agent, including an antibody or functional fragmentthereof, and a pharmaceutically acceptable carrier.

Still further embodiments of the invention include methods ofeffectively treating an animal suffering from an αVβ6-related disease ordisorder, including selecting an animal in need of treatment for aneoplastic or non-neoplastic disease, and administering to the animal atherapeutically effective dose of a fully human monoclonal antibody thatspecifically binds to αVβ6.

The antibodies of the invention can be used to treat an αVβ6-relateddisease or disorder. An αVβ6-related disease or disorder can be anycondition arising due to the aberrant activation or expression of αVβ6.Examples of such diseases include where αVβ6 aberrantly interacts withits ligands thereby altering cell-adhesion or cell signaling properties.This alteration in cell adhesion or cell signaling properties can resultin neoplastic diseases. Other αVβ6-related diseases or disorders includeinflammatory disorders, lung disease, diseases associated with fibrosisand any disease associated with dysregulated TGF-β.

In one example, the αVβ6-related disease is a neoplastic disease such asmelanoma, small cell lung cancer, non-small cell lung cancer, glioma,hepatocellular (liver) carcinoma, thyroid tumor, gastric (stomach)cancer, prostate cancer, breast cancer, ovarian cancer, bladder cancer,lung cancer, glioblastoma, endometrial cancer, kidney cancer, coloncancer, pancreatic cancer, esophageal carcinoma, head and neck cancers,mesothelioma, sarcomas, biliary (cholangiocarcinoma), small boweladenocarcinoma, pediatric malignancies and epidermoid carcinoma.

In another example, the αVβ6-related disease is an inflammatory disordersuch as inflammatory bowel disease; systemic lupus erythematosus;rheumatoid arthritis; juvenile chronic arthritis; spondyloarthropathies;systemic sclerosis, for example, scleroderma; idiopathic inflammatorymyopathies for example, dermatomyositis, polymyositis; Sjogren'ssyndrome; systemic vaculitis; sarcoidosis; thyroiditis, for example,Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis; immune-mediated renal disease, forexample, glomerulonephritis, tubulointerstitial nephritis; demyelinatingdiseases of the central and peripheral nervous systems such as multiplesclerosis, idiopathic polyneuropathy; hepatobiliary diseases such asinfectious hepatitis such as hepatitis A, B, C, D, E and othernonhepatotropic viruses; autoimmune chronic active hepatitis; primarybiliary cirrhosis; granulomatous hepatitis; and sclerosing cholangitis;inflammatory and fibrotic lung diseases (e.g., cystic fibrosis);gluten-sensitive enteropathy; autoimmune or immune-mediated skindiseases including bullous skin diseases, erythema multiforme andcontact dermatitis, psoriasis; allergic diseases of the lung such aseosinophilic pneumonia, idiopathic pulmonary fibrosis, allergicconjunctivitis and hypersensitivity pneumonitis, transplantationassociated diseases including graft rejection and graft-versus hostdisease.

In yet another example, the αVβ6-related disease is fibrosis such askidney or lung fibrosis.

In yet another example, the αVβ6-related disease is associated withdysregulated TGF-β include cancer and connective tissue (fibrotic)disorders.

Additional embodiments of the invention include methods of inhibitingαVβ6 induced cell adhesion in an animal. These methods include selectingan animal in need of treatment for αVβ6 induced cell adhesion, andadministering to said animal a therapeutically effective dose of a fullyhuman monoclonal antibody wherein said antibody specifically binds toαVβ6.

Further embodiments of the invention include the use of an antibody inthe preparation of medicament for the treatment of an αVβ6 relateddisease or disorder in an animal, wherein said monoclonal antibodyspecifically binds to αVβ6.

In still further embodiments, the targeted binding agents describedherein can be used for the preparation of a medicament for the effectivetreatment of αVβ6 induced cell adhesion in an animal, wherein saidmonoclonal antibody specifically binds to αVβ6.

Embodiments of the invention described herein relate to monoclonalantibodies that bind αVβ6 and affect αVβ6 function. Other embodimentsrelate to fully human anti-αVβ6 antibodies and anti-αVβ6 antibodypreparations with desirable properties from a therapeutic perspective,including high binding affinity for αVβ6, the ability to neutralize αVβ6in vitro and in vivo, and the ability to inhibit αVβ6 induced celladhesion and tumor growth.

In one embodiment, the invention includes antibodies that bind to αVβ6with very high affinities (KD). For example a human, rabbit, mouse,chimeric or humanized antibody that is capable of binding αVβ6 with a Kdless than, but not limited to, about 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰or about 10⁻¹¹M, or any range or value therein. Affinity and/or aviditymeasurements can be measured by KinExA® and/or BIACORE®, as describedherein.

One embodiment of the invention includes isolated antibodies, orfragments of those antibodies, that bind to αVβ6. As known in the art,the antibodies can be, for example, polyclonal, oligoclonal, monoclonal,chimeric, humanized, and/or fully human antibodies. Embodiments of theinvention described herein also provide cells for producing theseantibodies.

It will be appreciated that embodiments of the invention are not limitedto any particular form of an antibody or method of generation orproduction. For example, the anti-αVβ6 antibody may be a full-lengthantibody (e.g., having an intact human Fc region) or an antibodyfragment (e.g., a Fab, Fab′ or F(ab′)₂, FV or Dab (Dabs are the smallestfunctional binding units of human antibodies). In addition, the antibodymay be manufactured from a hybridoma that secretes the antibody, or froma recombinantly produced cell that has been transformed or transfectedwith a gene or genes encoding the antibody.

Other embodiments of the invention include isolated nucleic acidmolecules encoding any of the antibodies described herein, vectorshaving isolated nucleic acid molecules encoding anti-αVβ6 antibodies ora host cell transformed with any of such nucleic acid molecules. Inaddition, one embodiment of the invention is a method of producing ananti-αVβ6 antibody by culturing host cells under conditions wherein anucleic acid molecule is expressed to produce the antibody followed byrecovering the antibody. It should be realized that embodiments of theinvention also include any nucleic acid molecule which encodes anantibody or fragment of an antibody of the invention including nucleicacid sequences optimized for increasing yields of antibodies orfragments thereof when transfected into host cells for antibodyproduction.

A further embodiment includes a method of producing high affinityantibodies to αVβ6 by immunizing a mammal with human αVβ6, or a fragmentthereof, and one or more orthologous sequences or fragments thereof.

Other embodiments are based upon the generation and identification ofisolated antibodies that bind specifically to αVβ6. Inhibition of thebiological activity of αVβ6 can prevent αVβ6 induced cell adhesion andother desired effects.

Another embodiment of the invention includes a method of diagnosingdiseases or conditions in which an antibody prepared as described hereinis utilized to detect the level of αVβ6 in a patient sample. In oneembodiment, the patient sample is blood or blood serum. In furtherembodiments, methods for the identification of risk factors, diagnosisof disease, and staging of disease is presented which involves theidentification of the overexpression of αVβ6 using anti-αVβ6 antibodies.

Another embodiment of the invention includes a method for diagnosing acondition associated with the expression of αVβ6 in a cell by contactingthe serum or a cell with an anti-αVβ6 antibody, and thereafter detectingthe presence of αVβ6. Preferred conditions include an αVβ6 relateddisease or disorder including, but not limited to, neoplastic diseases,such as, melanoma, small cell lung cancer, non-small cell lung cancer,glioma, hepatocellular (liver) carcinoma, glioblastoma, and carcinoma ofthe thyroid, stomach, prostate, breast, ovary, bladder, lung, uterus,kidney, colon, and pancreas, salivary gland, and colorectum.

In another embodiment, the invention includes an assay kit for detectingαVβ6 in mammalian tissues, cells, or body fluids to screen forαVβ6-related diseases. The kit includes an antibody that binds to αVβ6and a means for indicating the reaction of the antibody with αVβ6, ifpresent. In one embodiment, the antibody is a monoclonal antibody. Inanother embodiment, the antibody that binds αVβ6 is labeled. In stillanother embodiment the antibody is an unlabeled primary antibody and thekit further includes a means for detecting the primary antibody. In oneembodiment, the means for detecting includes a labeled second antibodythat is an anti-immunoglobulin. The antibody may be labeled with amarker selected from the group consisting of a fluorochrome, an enzyme,a radionuclide and a radiopaque material.

Other embodiments of the invention include pharmaceutical compositionshaving an effective amount of an anti-αVβ6 antibody in admixture with apharmaceutically acceptable carrier or diluent. In yet otherembodiments, the anti-αVβ6 antibody, or a fragment thereof, isconjugated to a therapeutic agent. The therapeutic agent can be, forexample, a toxin or a radioisotope.

Yet another embodiment includes methods for treating diseases orconditions associated with the expression of αVβ6 in a patient, byadministering to the patient an effective amount of an anti-αVβ6antibody. The anti-αVβ6 antibody can be administered alone, or can beadministered in combination with additional antibodies orchemotherapeutic drug or radiation therapy. For example, a monoclonal,oligoclonal or polyclonal mixture of αVβ6 antibodies that block celladhesion can be administered in combination with a drug shown to inhibittumor cell proliferation directly. The method can be performed in vivoand the patient is preferably a human patient. In a preferredembodiment, the method concerns the treatment of an αVβ6 related diseaseor disorder including, but not limited to, neoplastic diseases, such as,melanoma, small cell lung cancer, non-small cell lung cancer, glioma,hepatocellular (liver) carcinoma, glioblastoma, and carcinoma of thethyroid, stomach, prostate, breast, ovary, bladder, lung, uterus,kidney, colon, and pancreas, salivary gland, and colorectum.

In another embodiment, the invention provides an article of manufactureincluding a container. The container includes a composition containingan anti-αVβ6 antibody, and a package insert or label indicating that thecomposition can be used to treat an αVβ6 related disease or disordercharacterized by the overexpression of αVβ6.

In some embodiments, the anti-αVβ6 antibody is administered to apatient, followed by administration of a clearing agent to remove excesscirculating antibody from the blood.

Yet another embodiment is the use of an anti-αVβ6 antibody in thepreparation of a medicament for the treatment of αVβ6-related diseasesor disorders such as neoplastic diseases, inflammatory disorders,fibrosis, lung disease or diseases associated with dysregulated TGF-β.In one embodiment, the neoplastic diseases include carcinoma, such asbreast, ovarian, stomach, endometrial, salivary gland, lung, kidney,colon, colorectum, esophageal, thyroid, pancreatic, prostate and bladdercancer. In another embodiment, the αVβ6 related diseases or disordersinclude, but are not limited to, neoplastic diseases, such as, melanoma,small cell lung cancer, non-small cell lung cancer, glioma,hepatocellular (liver) carcinoma, sarcoma, head and neck cancers,mesothelioma, biliary (cholangiocarcinoma), small bowel adenocarcinoma,pediatric malignancies and glioblastoma.

Yet another embodiment of the invention is the use of an anti-αVβ6antibody in the preparation of a medicament for the treatment ofinflammatory, or hyperprolifearative diseases including but not limitedto arthritis, atherosclerosis, allergic conjunctivitis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a line graph showing the ability of the purified monoclonalantibodies to bind to αVβ6 and block its binding to a GST-LAP peptide.

FIGS. 2A and 2B are line graphs showing a plot of the averaged GeometricMean Fluorescence (GMF) as a function of molecular mAb concentration,which was used to estimate the binding affinity of one of the antibodiesto K562 cells that stably express the human αVβ6 antigen. Shown in FIG.2A is affinity data for mAb 188. FIG. 2B shows affinity data for mAb 264RAD.

FIGS. 3A-3E are bar graphs showing the ability of the purifiedmonoclonal antibodies to mediate complement-dependent cytotoxicity in293 cells stably expressing αVβ6 integrin.

FIG. 4 is a bar graph showing the ability of antibodies 264RAD, 133 and188 SDM to inhibit tumour growth using the Detroit-562 nasophayngealcell line.

FIG. 5 is a bar chart showing comparison of the activity of 264 RAD with264 RAD/ADY.

DETAILED DESCRIPTION

Embodiments of the invention relate to targeted binding agents that bindto αVβ6 integrin (αVβ6). In some embodiments, the binding agents bind toαVβ6 and inhibit the binding of ligands to αVβ6. In one embodiment, thetargeted binding agents are monoclonal antibodies, or binding fragmentsthereof. In another embodiment, the antibodies bind only to the β6 chainyet are able to inhibit binding of ligands to αVβ6.

Other embodiments of the invention include fully human anti-αVβ6antibodies, and antibody preparations that are therapeutically useful.In one embodiment, the anti-αVβ6 antibody preparations have desirabletherapeutic properties, including strong binding affinity for αVβ6, andthe ability to inhibit TGFβLAP mediated cell adhesion in vitro.

Embodiments of the invention also include fully human isolated bindingfragments of anti-αVβ6 antibodies. In one embodiment the bindingfragments are derived from fully human anti-αVβ6 antibodies. Exemplaryfragments include Fv, Fab′ or other well-known antibody fragments, asdescribed in more detail below. Embodiments of the invention alsoinclude cells that express fully human antibodies against αVβ6. Examplesof cells include hybridomas, or recombinantly created cells, such asChinese hamster ovary (CHO) cells, variants of CHO cells (for exampleDG44) and NS0 cells that produce antibodies against αVβ6. Additionalinformation about variants of CHO cells can be found in Andersen andReilly (2004) Current Opinion in Biotechnology 15, 456-462 which isincorporated herein in its entirety by reference.

In addition, embodiments of the invention include methods of using theseantibodies for treating an αVβ6-related disease or disorder. AnαVβ6-related disease or disorder can be any condition arising due to theaberrant activation or expression of αVβ6. Examples of such diseasesinclude where αVβ6 aberrantly interacts with its ligands therebyaltering cell-adhesion or cell signaling properties. This alteration incell adhesion or cell signaling properties can result in neoplasticdiseases. Other αVβ6-related diseases or disorders include inflammatorydisorders, lung disease, diseases associated with fibrosis and anydisease associated with dysregulated TGF-β.

In one example, the αVβ6-related disease is a neoplastic disease such asmelanoma, small cell lung cancer, non-small cell lung cancer, glioma,hepatocellular (liver) carcinoma, thyroid tumor, gastric (stomach)cancer, prostate cancer, breast cancer, ovarian cancer, bladder cancer,lung cancer, glioblastoma, endometrial cancer, kidney cancer, coloncancer, pancreatic cancer, esophageal carcinoma, head and neck cancers,mesothelioma, sarcomas, biliary (cholangiocarcinoma), small boweladenocarcinoma, pediatric malignancies and epidermoid carcinoma.

In another example, the αVβ6-related disease is an inflammatory disordersuch as inflammatory bowel disease; systemic lupus erythematosus;rheumatoid arthritis; juvenile chronic arthritis; spondyloarthropathies;systemic sclerosis, for example, scleroderma; idiopathic inflammatorymyopathies for example, dermatomyositis, polymyositis; Sjogren'ssyndrome; systemic vaculitis; sarcoidosis; thyroiditis, for example,Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis; immune-mediated renal disease, forexample, glomerulonephritis, tubulointerstitial nephritis; demyelinatingdiseases of the central and peripheral nervous systems such as multiplesclerosis, idiopathic polyneuropathy; hepatobiliary diseases such asinfectious hepatitis such as hepatitis A, B, C, D, E and othernonhepatotropic viruses; autoimmune chronic active hepatitis; primarybiliary cirrhosis; granulomatous hepatitis; and sclerosing cholangitis;inflammatory and fibrotic lung diseases (e.g., cystic fibrosis);gluten-sensitive enteropathy; autoimmune or immune-mediated skindiseases including bullous skin diseases, erythema multiforme andcontact dermatitis, psoriasis; allergic diseases of the lung such aseosinophilic pneumonia, idiopathic pulmonary fibrosis, allergicconjunctivitis and hypersensitivity pneumonitis, transplantationassociated diseases including graft rejection and graft-versus hostdisease.

In yet another example, the αVβ6-related disease is fibrosis such askidney or lung fibrosis.

In yet another example, the αVβ6-related disease is associated withdysregulated TGF-β include cancer and connective tissue (fibrotic)disorders.

Other embodiments of the invention include diagnostic assays forspecifically determining the quantity of αVβ6 in a biological sample.The assay kit can include anti-αVβ6 antibodies along with the necessarylabels for detecting such antibodies. These diagnostic assays are usefulto screen for αV related diseases or β6 disorders including, but notlimited to, neoplastic diseases, such as, melanoma, small cell lungcancer, non-small cell lung cancer, glioma, hepatocellular (liver)carcinoma, glioblastoma, and carcinoma of the thyroid, stomach,prostate, breast, ovary, bladder, lung, uterus, kidney, colon, andpancreas, salivary gland, and colorectum.

Another aspect of the invention is an antagonist of the biologicalactivity of αVβ6 wherein the antagonist binds to αVβ6. In oneembodiment, the antagonist is a targeted binding agent, such as anantibody. The antagonist may bind to:

-   -   i) β6 alone;    -   ii) αVβ6; or    -   iii) the αVβ6/ligand complex,        or a combination of these. In one embodiment the antibody is        able to antagonize the biological activity of αVβ6 in vitro and        in vivo. The antibody may be selected from fully human        monoclonal antibody e.g., sc 264 RAD, sc 264 RAD/ADY, sc 188        SDM, sc 133, sc 133 TMT, sc 133 WDS, sc 133 TMT/WDS, sc 188, sc        254, sc 264 or sc 298 or variants thereof.

In one embodiment the antagonist of the biological activity of αVβ6 maybind to αVβ6 and thereby prevent TGFβLAP mediated cell adhesion.

One embodiment is an antibody which binds to the same epitope orepitopes as fully human monoclonal antibody c 264 RAD, sc 264 RAD/ADY,sc 188 SDM, sc 133, sc 133 TMT, sc 133 WDS, sc 133 TMT/WDS, sc 188, sc254, sc 264 or sc 298.

In one embodiment, the targeted binding agent binds αVβ6 with a K_(d) ofless than 100 nanomolar (nM). The targeted binding agent may bind with aK_(d) less than about 35 nanomolar (nM). The targeted binding agent maybind with a K_(d) less than about 25 nanomolar (nM). The targetedbinding agent may bind with a K_(d) less than about 10 nanomolar (nM).In another embodiment, the targeted binding agent binds with a K_(d) ofless than about 60 picomolar (pM).

One embodiment is an antibody-secreting plasma cell that produces thelight chain and/or the heavy chain of antibody as described hereinabove.In one embodiment the plasma cell produces the light chain and/or theheavy chain of a fully human monoclonal antibody. In another embodimentthe plasma cell produces the light chain and/or the heavy chain of thefully human monoclonal antibody c 264 RAD, sc 264 RAD/ADY, sc 188 SDM,sc 133, sc 133 TMT, sc 133 WDS, sc 133 TMT/WDS, sc 188, sc 254, sc 264or sc 298. Alternatively the plasma cell may produce an antibody whichbinds to the same epitope or epitopes as fully human monoclonal antibodysc c 264 RAD, sc 264 RAD/ADY, sc 188 SDM, sc 133, sc 133 TMT, sc 133WDS, sc 133 TMT/WDS, sc 188, sc 254, sc 264 or sc 298.

Another embodiment is a nucleic acid molecule encoding the light chainor the heavy chain of an antibody as described hereinabove. In oneembodiment the nucleic acid molecule encodes the light chain or theheavy chain of a fully human monoclonal antibody. Still anotherembodiment is a nucleic acid molecule encoding the light chain or theheavy chain of a fully human monoclonal antibody selected fromantibodies c 264 RAD, sc 264 RAD/ADY, sc 188 SDM, sc 133, sc 133 TMT, sc133 WDS, sc 133 TMT/WDS, sc 188, sc 254, sc 264 or sc 298.

Another embodiment of the invention is a vector comprising a nucleicacid molecule or molecules as described hereinabove, wherein the vectorencodes a light chain and/or a heavy chain of an antibody as definedhereinabove.

Yet another embodiment of the invention is a host cell comprising avector as described hereinabove. Alternatively the host cell maycomprise more than one vector.

In addition, one embodiment of the invention is a method of producing anantibody by culturing host cells under conditions wherein a nucleic acidmolecule is expressed to produce the antibody, followed by recovery ofthe antibody.

In one embodiment the invention includes a method of making an antibodyby transfecting at least one host cell with at least one nucleic acidmolecule encoding the antibody as described hereinabove, expressing thenucleic acid molecule in the host cell and isolating the antibody.

According to another aspect, the invention includes a method ofantagonising the biological activity of αVβ6 comprising administering anantagonist as described herein. The method may include selecting ananimal in need of treatment for an αVβ6 related disease or disorder, andadministering to the animal a therapeutically effective dose of anantagonist of the biological activity of αVβ6.

Another aspect of the invention includes a method of antagonising thebiological activity of αVβ6 comprising administering an antibody asdescribed hereinabove. The method may include selecting an animal inneed of treatment for an αVβ6 related disease or disorder, andadministering to said animal a therapeutically effective dose of anantibody which antagonises the biological activity of αVβ6.

According to another aspect there is provided a method of treating anαVβ6 related disease or disorder in a mammal comprising administering atherapeutically effective amount of an antagonist of the biologicalactivity of αVβ6. The method may include selecting an animal in need oftreatment for an αVβ6 related disease or disorder, and administering tosaid animal a therapeutically effective dose of an antagonist of thebiological activity of αVβ6.

According to another aspect there is provided a method of treating anαVβ6 disease or disorder in a mammal comprising administering atherapeutically effective amount of an antibody which antagonizes thebiological activity of αVβ6. The method may include selecting an animalin need of treatment for an αVβ6 related disease or disorder, andadministering to said animal a therapeutically effective dose of anantibody which antagonises the biological activity of αVβ6. The antibodycan be administered alone, or can be administered in combination withadditional antibodies or chemotherapeutic drug or radiation therapy.

According to another aspect there is provided a method of treatingcancer in a mammal comprising administering a therapeutically effectiveamount of an antagonist of the biological activity of αVβ6. The methodmay include selecting an animal in need of treatment for cancer, andadministering to said animal a therapeutically effective dose of anantagonist which antagonises the biological activity of αVβ6. Theantagonist can be administered alone, or can be administered incombination with additional antibodies or chemotherapeutic drug orradiation therapy.

According to another aspect there is provided a method of treatingcancer in a mammal comprising administering a therapeutically effectiveamount of an antibody which antagonizes the biological activity of αVβ6.The method may include selecting an animal in need of treatment forcancer, and administering to said animal a therapeutically effectivedose of an antibody which antagonises the biological activity of αVβ6.The antibody can be administered alone, or can be administered incombination with additional antibodies or chemotherapeutic drug orradiation therapy.

According to another aspect of the invention there is provided the useof an antagonist of the biological activity of αVβ6 for the manufactureof a medicament for the treatment of an αVβ6 related disease ordisorder.

According to another aspect of the invention there is provided the useof an antibody which antagonizes the biological activity of αVβ6 for themanufacture of a medicament for the treatment of an αVβ6 related diseaseor disorder.

In a preferred embodiment the present invention is particularly suitablefor use in antagonizing αVβ6, in patients with a tumor which isdependent alone, or in part, on αVβ6 integrin.

Another embodiment of the invention includes an assay kit for detectingαVβ6 in mammalian tissues, cells, or body fluids to screen for an αVβ6related disease or disorder. The kit includes an antibody that binds toαVβ6 and a means for indicating the reaction of the antibody with αVβ6,if present. The antibody may be a monoclonal antibody. In oneembodiment, the antibody that binds αVβ6 is labeled. In anotherembodiment the antibody is an unlabeled primary antibody and the kitfurther includes a means for detecting the primary antibody. In oneembodiment, the means includes a labeled second antibody that is ananti-immunoglobulin. Preferably the antibody is labeled with a markerselected from the group consisting of a fluorochrome, an enzyme, aradionuclide and a radio-opaque material.

Further embodiments, features, and the like regarding anti-αVβ6antibodies are provided in additional detail below.

Sequence Listing

Embodiments of the invention include the specific anti-αVβ6 antibodieslisted below in Table 1. This table reports the identification number ofeach anti-αVβ6 antibody, along with the SEQ ID number of the variabledomain of the corresponding heavy chain and light chain genes. Eachantibody has been given an identification number that includes theletters “sc” followed by a number.

TABLE 1 Sequence mAb ID No.: NO: SEQ ID sc 49 Nucleotide sequenceencoding the variable region of the heavy chain 1 Amino acid sequenceencoding the variable region of the heavy chain 2 Nucleotide sequenceencoding the variable region of the light chain 3 Amino acid sequenceencoding the variable region of the light chain 4 sc 58 Nucleotidesequence encoding the variable region of the heavy chain 5 Amino acidsequence encoding the variable region of the heavy chain 6 Nucleotidesequence encoding the variable region of the light chain 7 Amino acidsequence encoding the variable region of the light chain 8 sc 97Nucleotide sequence encoding the variable region of the heavy chain 9Amino acid sequence encoding the variable region of the heavy chain 10Nucleotide sequence encoding the variable region of the light chain 11Amino acid sequence encoding the variable region of the light chain 12sc 133 Nucleotide sequence encoding the variable region of the heavychain 13 Amino acid sequence encoding the variable region of the heavychain 14 Nucleotide sequence encoding the variable region of the lightchain 15 Amino acid sequence encoding the variable region of the lightchain 16 sc 161 Nucleotide sequence encoding the variable region of theheavy chain 17 Amino acid sequence encoding the variable region of theheavy chain 18 Nucleotide sequence encoding the variable region of thelight chain 19 Amino acid sequence encoding the variable region of thelight chain 20 sc 188 Nucleotide sequence encoding the variable regionof the heavy chain 21 Amino acid sequence encoding the variable regionof the heavy chain 22 Nucleotide sequence encoding the variable regionof the light chain 23 Amino acid sequence encoding the variable regionof the light chain 24 sc 254 Nucleotide sequence encoding the variableregion of the heavy chain 25 Amino acid sequence encoding the variableregion of the heavy chain 26 Nucleotide sequence encoding the variableregion of the light chain 27 Amino acid sequence encoding the variableregion of the light chain 28 sc 264 Nucleotide sequence encoding thevariable region of the heavy chain 29 Amino acid sequence encoding thevariable region of the heavy chain 30 Nucleotide sequence encoding thevariable region of the light chain 31 Amino acid sequence encoding thevariable region of the light chain 32 sc 277 Nucleotide sequenceencoding the variable region of the heavy chain 33 Amino acid sequenceencoding the variable region of the heavy chain 34 Nucleotide sequenceencoding the variable region of the light chain 35 Amino acid sequenceencoding the variable region of the light chain 36 sc 298 Nucleotidesequence encoding the variable region of the heavy chain 37 Amino acidsequence encoding the variable region of the heavy chain 38 Nucleotidesequence encoding the variable region of the light chain 39 Amino acidsequence encoding the variable region of the light chain 40 sc 320Nucleotide sequence encoding the variable region of the heavy chain 41Amino acid sequence encoding the variable region of the heavy chain 42Nucleotide sequence encoding the variable region of the light chain 43Amino acid sequence encoding the variable region of the light chain 44sc 374 Nucleotide sequence encoding the variable region of the heavychain 45 Amino acid sequence encoding the variable region of the heavychain 46 Nucleotide sequence encoding the variable region of the lightchain 47 Amino acid sequence encoding the variable region of the lightchain 48 sc 188 Nucleotide sequence encoding the variable region of theheavy chain 70 SDM Amino acid sequence encoding the variable region ofthe heavy chain 71 Nucleotide sequence encoding the variable region ofthe light chain 72 Amino acid sequence encoding the variable region ofthe light chain 73 sc 264 Nucleotide sequence encoding the variableregion of the heavy chain 74 RAD Amino acid sequence encoding thevariable region of the heavy chain 75 Nucleotide sequence encoding thevariable region of the light chain 76 Amino acid sequence encoding thevariable region of the light chain 77 sc 133 Nucleotide sequenceencoding the variable region of the heavy chain 78 TMT Amino acidsequence encoding the variable region of the heavy chain 79 Nucleotidesequence encoding the variable region of the light chain 80 Amino acidsequence encoding the variable region of the light chain 81 sc 133Nucleotide sequence encoding the variable region of the heavy chain 82WDS Amino acid sequence encoding the variable region of the heavy chain83 Nucleotide sequence encoding the variable region of the light chain84 Amino acid sequence encoding the variable region of the light chain85 sc 133 Nucleotide sequence encoding the variable region of the heavychain 86 TMT/ Amino acid sequence encoding the variable region of theheavy chain 87 WDS Nucleotide sequence encoding the variable region ofthe light chain 88 Amino acid sequence encoding the variable region ofthe light chain 89 sc 264 Nucleotide sequence encoding the variableregion of the heavy chain 90 ADY Amino acid sequence encoding thevariable region of the heavy chain 91 Nucleotide sequence encoding thevariable region of the light chain 92 Amino acid sequence encoding thevariable region of the light chain 93 sc 264 Nucleotide sequenceencoding the variable region of the heavy chain 94 RAD/A Amino acidsequence encoding the variable region of the heavy chain 95 DYNucleotide sequence encoding the variable region of the light chain 96Amino acid sequence encoding the variable region of the light chain 97Definitions

Unless otherwise defined, scientific and technical terms used hereinshall have the meanings that are commonly understood by those ofordinary skill in the art. Further, unless otherwise required bycontext, singular terms shall include pluralities and plural terms shallinclude the singular. Generally, nomenclatures utilized in connectionwith, and techniques of, cell and tissue culture, molecular biology, andprotein and oligo- or polynucleotide chemistry and hybridizationdescribed herein are those well known and commonly used in the art.

Standard techniques are used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques areperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification. See e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2001)), which is incorporated herein by reference.The nomenclatures utilized in connection with, and the laboratoryprocedures and techniques of, analytical chemistry, synthetic organicchemistry, and medicinal and pharmaceutical chemistry described hereinare those well known and commonly used in the art. Standard techniquesare used for chemical syntheses, chemical analyses, pharmaceuticalpreparation, formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

The term “and/or” as used herein is to be taken as specific disclosureof each of the two specified features or components with or without theother. For example “A and/or B” is to be taken as specific disclosure ofeach of (i) A, (ii) B and (iii) A and B, just as if each is set outindividually herein.

An antagonist may be a polypeptide, nucleic acid, carbohydrate, lipid,small molecular weight compound, an oligonucleotide, an oligopeptide,RNA interference (RNAi), antisense, a recombinant protein, an antibody,or conjugates or fusion proteins thereof. For a review of RNAi seeMilhavet O, Gary D S, Mattson M P. (Pharmacol Rev. 2003 December;55(4):629-48. Review.) and antisense see Opalinska J B, Gewirtz A M.(Sci STKE. 2003 Oct. 28; 2003 (206):pe47.)

Disease-related aberrant activation or expression of “αVβ6” may be anyabnormal, undesirable or pathological cell adhesion, for exampletumor-related cell adhesion. Cell adhesion-related diseases include, butare not limited to, non-solid tumors such as leukemia, multiple myelomaor lymphoma, and also solid tumors such as melanoma, small cell lungcancer, non-small cell lung cancer, glioma, hepatocellular (liver)carcinoma, glioblastoma, carcinoma of the thyroid, bile duct, bone,gastric, brain/CNS, head and neck, hepatic system, stomach, prostate,breast, renal, testicle, ovary, skin, cervix, lung, muscle, neuron,oesophageal, bladder, lung, uterus, vulva, endometrium, kidney,colorectum, pancreas, pleural/peritoneal membranes, salivary gland, andepidermous.

A compound refers to any small molecular weight compound with amolecular weight of less than about 2000 Daltons.

The term “αVβ6” refers to the heterodimer integrin molecule consistingof an αV chain and a β6 chain.

The term “neutralizing” when referring to a targeted binding agent, suchas an antibody, relates to the ability of said targeted binding agent toeliminate, or significantly reduce, the activity of a target antigen.Accordingly, a “neutralizing” anti-αVβ6 antibody is capable ofeliminating or significantly reducing the activity of αVβ6. Aneutralizing αVβ6 antibody may, for example, act by blocking the bindingof TGFβLAP to the integrin αVβ6. By blocking this binding, αVβ6 mediatedcell adhesion is significantly, or completely, eliminated. Ideally, aneutralizing antibody against αVβ6 inhibits cell adhesion.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide that has been isolated from its naturally occurringenvironment. Such polynucleotides may be genomic, cDNA, or synthetic.Isolated polynucleotides preferably are not associated with all or aportion of the polynucleotides they associate with in nature. Theisolated polynucleotides may be operably linked to anotherpolynucleotide that it is not linked to in nature. In addition, isolatedpolynucleotides preferably do not occur in nature as part of a largersequence.

The term “isolated protein” referred to herein means a protein that hasbeen isolated from its naturally occurring environment. Such proteinsmay be derived from genomic DNA, cDNA, recombinant DNA, recombinant RNA,or synthetic origin or some combination thereof, which by virtue of itsorigin, or source of derivation, the “isolated protein” (1) is notassociated with proteins found in nature, (2) is free of other proteinsfrom the same source, e.g. free of murine proteins, (3) is expressed bya cell from a different species, or (4) does not occur in nature.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein, fragments, and analogs are species of the polypeptidegenus. Preferred polypeptides in accordance with the invention comprisethe human heavy chain immunoglobulin molecules and the human kappa lightchain immunoglobulin molecules, as well as antibody molecules formed bycombinations comprising the heavy chain immunoglobulin molecules withlight chain immunoglobulin molecules, such as the kappa or lambda lightchain immunoglobulin molecules, and vice versa, as well as fragments andanalogs thereof. Preferred polypeptides in accordance with the inventionmay also comprise solely the human heavy chain immunoglobulin moleculesor fragments thereof.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory orotherwise is naturally-occurring.

The term “operably linked” as used herein refers to positions ofcomponents so described that are in a relationship permitting them tofunction in their intended manner. For example, a control sequence“operably linked” to a coding sequence is connected in such a way thatexpression of the coding sequence is achieved under conditionscompatible with the control sequences.

The term “polynucleotide” as referred to herein means a polymeric formof nucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide, orRNA-DNA hetero-duplexes. The term includes single and double strandedforms of DNA.

The term “oligonucleotide” referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring linkages. Oligonucleotides are apolynucleotide subset generally comprising a length of 200 bases orfewer. Preferably, oligonucleotides are 10 to 60 bases in length andmost preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases inlength. Oligonucleotides are usually single stranded, e.g. for probes;although oligonucleotides may be double stranded, e.g. for use in theconstruction of a gene mutant. Oligonucleotides can be either sense orantisense oligonucleotides.

The term “naturally occurring nucleotides” referred to herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate,phosphoroamidate, and the like. See e.g., LaPlanche et al., Nucl. AcidsRes. 14:9081 (1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984);Stein et al., Nucl. Acids Res. 16:3209 (1988); Zon et al., Anti-CancerDrug Design 6:539 (1991); Zon et al., Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al., U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990), the disclosures ofwhich are hereby incorporated by reference. An oligonucleotide caninclude a label for detection, if desired.

The term “selectively hybridize” referred to herein means to detectablyand specifically bind. Polynucleotides, oligonucleotides and fragmentsthereof selectively hybridize to nucleic acid strands underhybridization and wash conditions that minimize appreciable amounts ofdetectable binding to nonspecific nucleic acids. High stringencyconditions can be used to achieve selective hybridization conditions asknown in the art and discussed herein. Generally, the nucleic acidsequence homology between the polynucleotides, oligonucleotides, orantibody fragments and a nucleic acid sequence of interest will be atleast 80%, and more typically with preferably increasing homologies ofat least 85%, 90%, 95%, 99%, and 100%.

The term “CDR region” or “CDR” is intended to indicate the hypervariableregions of the heavy and light chains of the immunoglobulin as definedby Kabat et al., 1991 (Kabat, E. A. et al., (1991) Sequences of Proteinsof Immunological Interest, 5th Edition. US Department of Health andHuman Services, Public Service, NIH, Washington), and later editions. Anantibody typically contains 3 heavy chain CDRs and 3 light chain CDRs.The term CDR or CDRs is used here in order to indicate, according to thecase, one of these regions or several, or even the whole, of theseregions which contain the majority of the amino acid residuesresponsible for the binding by affinity of the antibody for the antigenor the epitope which it recognizes.

Among the six short CDR sequences, the third CDR of the heavy chain(HCDR3) has a greater size variability (greater diversity essentiallydue to the mechanisms of arrangement of the genes which give rise toit). It may be as short as 2 amino acids although the longest size knownis 26. CDR length may also vary according to the length that can beaccommodated by the particular underlying framework. Functionally, HCDR3plays a role in part in the determination of the specificity of theantibody (Segal et al., PNAS, 71:4298-4302, 1974, Amit et al., Science,233:747-753, 1986, Chothia et al., J. Mol. Biol., 196:901-917, 1987,Chothia et al., Nature, 342:877-883, 1989, Caton et al., J. Immunol.,144:1965-1968, 1990, Sharon et al., PNAS, 87:4814-4817, 1990, Sharon etal., J. Immunol., 144:4863-4869, 1990, Kabat et al., J. Immunol.,147:1709-1719, 1991).

The term a “set of CDRs” referred to herein comprises CDR1, CDR2 andCDR3. Thus, a set of HCDRs refers to HCDR1, HCDR2 and HCDR3 (HCDR refersto a variable heavy chain CDR), and a set of LCDRs refers to LCDR1,LCDR2 and LCDR3 (LCDR refers to a variable light chain CDR). Unlessotherwise stated, a “set of CDRs” includes HCDRs and LCDRs.

Two amino acid sequences are “homologous” if there is a partial orcomplete identity between their sequences. For example, 85% homologymeans that 85% of the amino acids are identical when the two sequencesare aligned for maximum matching. Gaps (in either of the two sequencesbeing matched) are allowed in maximizing matching; gap lengths of 5 orless are preferred with 2 or less being more preferred. Alternativelyand preferably, two protein sequences (or polypeptide sequences derivedfrom them of at least about 30 amino acids in length) are homologous, asthis term is used herein, if they have an alignment score of at morethan 5 (in standard deviation units) using the program ALIGN with themutation data matrix and a gap penalty of 6 or greater. See Dayhoff, M.O., in Atlas of Protein Sequence and Structure, pp. 101-110 (Volume 5,National Biomedical Research Foundation (1972)) and Supplement 2 to thisvolume, pp. 1-10. The two sequences or parts thereof are more preferablyhomologous if their amino acids are greater than or equal to 50%identical when optimally aligned using the ALIGN program. It should beappreciated that there can be differing regions of homology within twoorthologous sequences. For example, the functional sites of mouse andhuman orthologues may have a higher degree of homology thannon-functional regions.

The term “corresponds to” is used herein to mean that a polynucleotidesequence is homologous (i.e., is identical, not strictly evolutionarilyrelated) to all or a portion of a reference polynucleotide sequence, orthat a polypeptide sequence is identical to a reference polypeptidesequence.

In contradistinction, the term “complementary to” is used herein to meanthat the complementary sequence is homologous to all or a portion of areference polynucleotide sequence. For illustration, the nucleotidesequence “TATAC” corresponds to a reference sequence “TATAC” and iscomplementary to a reference sequence “GTATA.”

The term “sequence identity” means that two polynucleotide or amino acidsequences are identical (i.e., on a nucleotide-by-nucleotide orresidue-by-residue basis) over the comparison window. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) or amino acid residue occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the comparison window(i.e., the window size), and multiplying the result by 100 to yield thepercentage of sequence identity. The terms “substantial identity” asused herein denotes a characteristic of a polynucleotide or amino acidsequence, wherein the polynucleotide or amino acid comprises a sequencethat has at least 85 percent sequence identity, preferably at least 90to 95 percent sequence identity, more preferably at least 99 percentsequence identity, as compared to a reference sequence over a comparisonwindow of at least 18 nucleotide (6 amino acid) positions, frequentlyover a window of at least 24-48 nucleotide (8-16 amino acid) positions,wherein the percentage of sequence identity is calculated by comparingthe reference sequence to the sequence which may include deletions oradditions which total 20 percent or less of the reference sequence overthe comparison window. The reference sequence may be a subset of alarger sequence.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis(2^(nd) Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), which is incorporated herein by reference.Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α-, α-disubstituted amino acids,N-alkyl amino acids, lactic acid, and other unconventional amino acidsmay also be suitable components for polypeptides of the presentinvention. Examples of unconventional amino acids include:4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and othersimilar amino acids and imino acids (e.g., 4-hydroxyproline). In thepolypeptide notation used herein, the left-hand direction is the aminoterminal direction and the right-hand direction is the carboxy-terminaldirection, in accordance with standard usage and convention.

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end; the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction; sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”; sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences”.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity, and mostpreferably at least 99 percent sequence identity. Preferably, residuepositions that are not identical differ by conservative amino acidsubstitutions. Conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. For example,a group of amino acids having aliphatic side chains is glycine, alanine,valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamic-aspartic, and asparagine-glutamine.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid sequence maintain at least about 75%, more preferably atleast 80%, 90%, 95%, and most preferably about 99% sequence identity tothe antibodies or immunoglobulin molecules described herein. Inparticular, conservative amino acid replacements are contemplated.Conservative replacements are those that take place within a family ofamino acids that have related side chains. Genetically encoded aminoacids are generally divided into families: (1) acidic=aspartate,glutamate; (2) basic=lysine, arginine, histidine; (3) non-polar=alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine,cysteine, serine, threonine, tyrosine. More preferred families are:serine and threonine are an aliphatic-hydroxy family; asparagine andglutamine are an amide-containing family; alanine, valine, leucine andisoleucine are an aliphatic family; and phenylalanine, tryptophan, andtyrosine are an aromatic family. For example, it is reasonable to expectthat an isolated replacement of a leucine with an isoleucine or valine,an aspartate with a glutamate, a threonine with a serine, or a similarreplacement of an amino acid with a structurally related amino acid willnot have a major effect on the binding function or properties of theresulting molecule, especially if the replacement does not involve anamino acid within a framework site.

Whether an amino acid change results in a functional peptide can readilybe determined by assaying the specific activity of the polypeptidederivative. Assays are described in detail herein. Fragments or analogsof antibodies or immunoglobulin molecules can be readily prepared bythose of ordinary skill in the art. Preferred amino- and carboxy-terminiof fragments or analogs occur near boundaries of functional domains.Structural and functional domains can be identified by comparison of thenucleotide and/or amino acid sequence data to public or proprietarysequence databases. Preferably, computerized comparison methods are usedto identify sequence motifs or predicted protein conformation domainsthat occur in other proteins of known structure and/or function. Methodsto identify protein sequences that fold into a known three-dimensionalstructure are known. Bowie et al., (1991) Science 253:164. Thus, theforegoing examples demonstrate that those of skill in the art canrecognize sequence motifs and structural conformations that may be usedto define structural and functional domains in accordance with theantibodies described herein.

A further aspect of the invention is a targeting binding agent or anantibody molecule comprising a VH domain that has at least about 60, 70,80, 85, 90, 95, 98 or about 99% amino acid sequence identity with a VHdomain of any of antibodies shown in Table 1, the appended sequencelisting, an antibody described herein, or with an HCDR (e.g., HCDR1,HCDR2, or HCDR3) shown in Table 8 or Table 29. The targeting bindingagent or antibody molecule may optionally also comprise a VL domain thathas at least about 60, 70, 80, 85, 90, 95, 98 or about 99% amino acidsequence identity with a VL domain any of antibodies shown in Table 1,the appended sequence listing, an antibody described herein, or with anLCDR (e.g., LCDR1, LCDR2, or LCDR3) shown in Table 9 or Table 30.Algorithms that can be used to calculate % identity of two amino acidsequences comprise e.g. BLAST (Altschul et al., (1990) J. Mol. Biol.215: 405-410), FASTA (Pearson and Lipman (1988) PNAS USA 85: 2444-2448),or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol.147: 195-197), e.g. employing default parameters. In some embodiments,the targeting binding agent or antibody that shares amino acid sequenceidentity as describes above, exhibits substantially the same activity asthe antibodies referenced. For instance, substantially the same activitycomprises at least one activity that differed from the activity of thereferences antibodies by no more that about 50%, 40%, 30%, 20%, 10%, 5%,2%, 1% or less.

An antigen binding site is generally formed by the variable heavy (VH)and variable light (VL) immunoglobulin domains, with the antigen-bindinginterface formed by six surface polypeptide loops, termedcomplimentarity determining regions (CDRs). There are three CDRs in eachVH (HCDR1, HCDR2, HCDR3) and in each VL (LCDR1, LCDR2, LCDR3), togetherwith framework regions (FRs).

Typically, a VH domain is paired with a VL domain to provide an antibodyantigen-binding site, although a VH or VL domain alone may be used tobind antigen. The VH domain (e.g. from Table 1) may be paired with theVL domain (e.g. from Table 1), so that an antibody antigen-binding siteis formed comprising both the VH and VL domains. Analogous embodimentsare provided for the other VH and VL domains disclosed herein. In otherembodiments, VH chains in Table 8 or Table 29 are paired with aheterologous VL domain. Light-chain promiscuity is well established inthe art. Again, analogous embodiments are provided by the invention forthe other VH and VL domains disclosed herein. Thus, the VH of the parentor of any of antibodies chain on Table 9 or Table 30 may be paired withthe VL of the parent or of any of antibodies on Table 1 or otherantibody.

An antigen binding site may comprise a set of H and/or L CDRs of theparent antibody or any of antibodies in Table 1 with as many as twenty,sixteen, ten, nine or fewer, e.g. one, two, three, four or five, aminoacid additions, substitutions, deletions, and/or insertions within thedisclosed set of H and/or L CDRs. Alternatively, an antigen binding sitemay comprise a set of H and/or L CDRs of the parent antibody or any ofantibodies Table 1 with as many as twenty, sixteen, ten, nine or fewer,e.g. one, two, three, four or five, amino acid substitutions within thedisclosed set of H and/or L CDRs. Such modifications may potentially bemade at any residue within the set of CDRs.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (4) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et at. Nature 354:105 (1991), which are each incorporatedherein by reference.

A further aspect of the invention is an antibody molecule comprising aVH domain that has at least about 60, 70, 80, 85, 90, 95, 98 or about99% amino acid sequence identity with a VH domain of any of antibodieslisted in Table 1, the appended sequence listing or described herein, orwith an HCDR (e.g., HCDR1, HCDR2, or HCDR3) shown in Table 8 or Table29. The antibody molecule may optionally also comprise a VL domain thathas at least 60, 70, 80, 85, 90, 95, 98 or 99% amino acid sequenceidentity with a VL domain of any of the antibodies shown in Table 1, theappended sequence listing or described herein, or with an LCDR (e.g.,LCDR1, LCDR2, or LCDR3) shown in Table 9 or Table 30. Algorithms thatcan be used to calculate % identity of two amino acid sequences comprisee.g. BLAST (Altschul et al., (1990) J. Mol. Biol. 215: 405-410), FASTA(Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or theSmith-Waterman algorithm (Smith and Waterman (1981) J. Mol. Biol. 147:195-197), e.g. employing default parameters.

Variants of the VH and VL domains and CDRs of the present invention,including those for which amino acid sequences are set out herein, andwhich can be employed in targeting agents and antibodies for αVβ6 can beobtained by means of methods of sequence alteration or mutation andscreening for antigen targeting with desired characteristics. Examplesof desired characteristics include but are not limited to: increasedbinding affinity for antigen relative to known antibodies which arespecific for the antigen; increased neutralization of an antigenactivity relative to known antibodies which are specific for the antigenif the activity is known; specified competitive ability with a knownantibody or ligand to the antigen at a specific molar ratio; ability toimmunoprecipitate complex; ability to bind to a specified epitope;linear epitope, e.g. peptide sequence identified using peptide-bindingscan as described herein, e.g. using peptides screened in linear and/orconstrained conformation; conformational epitope, formed bynon-continuous residues; ability to modulate a new biological activityof αVβ6, or downstream molecule. Such methods are also provided herein.

Variants of antibody molecules disclosed herein may be produced and usedin the present invention. Following the lead of computational chemistryin applying multivariate data analysis techniques to thestructure/property-activity relationships (Wold, et al., Multivariatedata analysis in chemistry. Chemometrics—Mathematics and Statistics inChemistry (Ed.: B. Kowalski), D. Reidel Publishing Company, Dordrecht,Holland, 1984) quantitative activity-property relationships ofantibodies can be derived using well-known mathematical techniques, suchas statistical regression, pattern recognition and classification(Norman et al., Applied Regression Analysis. Wiley-Interscience; 3rdedition (April 1998); Kandel, Abraham & Backer, Eric. Computer-AssistedReasoning in Cluster Analysis. Prentice Hall PTR, (May 11, 1995);Krzanowski, Wojtek. Principles of Multivariate Analysis: A User'sPerspective (Oxford Statistical Science Series, No 22 (Paper)). OxfordUniversity Press; (December 2000); Witten, Ian H. & Frank, Eibe. DataMining: Practical Machine Learning Tools and Techniques with JavaImplementations. Morgan Kaufmann; (Oct. 11, 1999); Denison David G. T.(Editor), Christopher C. Holmes, Bani K. Mallick, Adrian F. M. Smith.Bayesian Methods for Nonlinear Classification and Regression (WileySeries in Probability and Statistics). John Wiley & Sons; (July 2002);Ghose, Arup K. & Viswanadhan, Vellarkad N. Combinatorial Library Designand Evaluation Principles, Software, Tools, and Applications in DrugDiscovery). The properties of antibodies can be derived from empiricaland theoretical models (for example, analysis of likely contact residuesor calculated physicochemical property) of antibody sequence, functionaland three-dimensional structures and these properties can be consideredsingly and in combination.

An antibody antigen-binding site composed of a VH domain and a VL domainis typically formed by six loops of polypeptide: three from the lightchain variable domain (VL) and three from the heavy chain variabledomain (VH). Analysis of antibodies of known atomic structure haselucidated relationships between the sequence and three-dimensionalstructure of antibody combining sites. These relationships imply that,except for the third region (loop) in VH domains, binding site loopshave one of a small number of main-chain conformations: canonicalstructures. The canonical structure formed in a particular loop has beenshown to be determined by its size and the presence of certain residuesat key sites in both the loop and in framework regions.

This study of sequence-structure relationship can be used for predictionof those residues in an antibody of known sequence, but of an unknownthree-dimensional structure, which are important in maintaining thethree-dimensional structure of its CDR loops and hence maintain bindingspecificity. These predictions can be backed up by comparison of thepredictions to the output from lead optimization experiments. In astructural approach, a model can be created of the antibody moleculeusing any freely available or commercial package, such as WAM. A proteinvisualisation and analysis software package, such as Insight II(Accelrys, Inc.) or Deep View may then be used to evaluate possiblesubstitutions at each position in the CDR. This information may then beused to make substitutions likely to have a minimal or beneficial effecton activity.

The techniques required to make substitutions within amino acidsequences of CDRs, antibody VH or VL domains and/or binding agentsgenerally are available in the art. Variant sequences may be made, withsubstitutions that may or may not be predicted to have a minimal orbeneficial effect on activity, and tested for ability to bind and/orneutralize and/or for any other desired property.

Variable domain amino acid sequence variants of any of the VH and VLdomains whose sequences are specifically disclosed herein may beemployed in accordance with the present invention, as discussed.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence deduced, for example, froma full-length cDNA sequence. Fragments typically are at least about 5,6, 8 or 10 amino acids long, preferably at least about 14 amino acidslong, more preferably at least about 20 amino acids long, usually atleast about 50 amino acids long, and even more preferably at least about70 amino acids long. The term “analog” as used herein refers topolypeptides which are comprised of a segment of at least about 25 aminoacids that has substantial identity to a portion of a deduced amino acidsequence and which has at least one of the following properties: (1)specific binding to αVβ6, under suitable binding conditions, (2) abilityto block appropriate ligand/αVβ6 binding, or (3) ability to inhibit αVβ6activity. Typically, polypeptide analogs comprise a conservative aminoacid substitution (or addition or deletion) with respect to thenaturally-occurring sequence. Analogs typically are at least 20 aminoacids long, preferably at least 50 amino acids long or longer, and canoften be as long as a full-length naturally-occurring polypeptide.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics.” Fauchere, J. Adv. Drug Res. 15:29(1986); Veber and Freidinger TINS p. 392 (1985); and Evans et al., J.Med. Chem. 30:1229 (1987), which are incorporated herein by reference.Such compounds are often developed with the aid of computerizedmolecular modeling. Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce an equivalenttherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to a paradigm polypeptide (i.e., a polypeptide thathas a biochemical property or pharmacological activity), such as humanantibody, but have one or more peptide linkages optionally replaced by alinkage selected from the group consisting of: —CH₂NH—, —CH₂S—,—CH₂—CH₂—, —CH═CH-(cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—, bymethods well known in the art. Systematic substitution of one or moreamino acids of a consensus sequence with a D-amino acid of the same type(e.g., D-lysine in place of L-lysine) may be used to generate morestable peptides. In addition, constrained peptides comprising aconsensus sequence or a substantially identical consensus sequencevariation may be generated by methods known in the art (Rizo andGierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference); for example, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclize the peptide.

As used herein, the term “antibody” refers to a polypeptide or group ofpolypeptides that are comprised of at least one binding domain that isformed from the folding of polypeptide chains having three-dimensionalbinding spaces with internal surface shapes and charge distributionscomplementary to the features of an antigenic determinant of an antigen.An antibody typically has a tetrameric form, comprising two identicalpairs of polypeptide chains, each pair having one “light” and one“heavy” chain. The variable regions of each light/heavy chain pair forman antibody binding site.

As used herein, a “targeted binding agent” is an agent, e.g. antibody,or binding fragment thereof, that preferentially binds to a target site.In one embodiment, the targeted binding agent is specific for only onetarget site. In other embodiments, the targeted binding agent isspecific for more than one target site. In one embodiment, the targetedbinding agent may be a monoclonal antibody and the target site may be anepitope. As described below, a targeted binding agent may comprise atleast one antigen binding domain of an antibody, wherein said domain isfused or contained within a heterologous protein.

“Binding fragments” of an antibody are produced by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intact antibodies.Binding fragments include Fab, Fab′, F(ab′)₂, Fv, and single-chainantibodies. An antibody other than a “bispecific” or “bifunctional”antibody is understood to have each of its binding sites identical. Anantibody substantially inhibits adhesion of a receptor to acounter-receptor when an excess of antibody reduces the quantity ofreceptor bound to counter-receptor by at least about 20%, 40%, 60% or80%, and more usually greater than about 85% (as measured in an in vitrocompetitive binding assay).

An antibody may be oligoclonal, a polyclonal antibody, a monoclonalantibody, a chimeric antibody, a CDR-grafted antibody, a multi-specificantibody, a bi-specific antibody, a catalytic antibody, a chimericantibody, a humanized antibody, a fully human antibody, ananti-idiotypic antibody and antibodies that can be labeled in soluble orbound form as well as fragments, variants or derivatives thereof, eitheralone or in combination with other amino acid sequences provided byknown techniques. An antibody may be from any species. The term antibodyalso includes binding fragments of the antibodies of the invention;exemplary fragments include Fv, Fab, Fab′, single stranded antibody(svFC), dimeric variable region (Diabody) and disulphide stabilizedvariable region (dsFv).

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (Ward,E. S. et al., (1989) Nature 341, 544-546) the Fab fragment consisting ofVL, VH, CL and CH1 domains; (McCafferty et al., (1990) Nature, 348,552-554) the Fd fragment consisting of the VH and CH1 domains; (Holt etal., (2003) Trends in Biotechnology 21, 484-490) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment (Ward, E. S. et al., Nature 341, 544-546 (1989), McCafferty etal., (1990) Nature, 348, 552-554, Holt et al., (2003) Trends inBiotechnology 21, 484-490], which consists of a VH or a VL domain; (v)isolated CDR regions; (vi) F(ab′)2 fragments, a bivalent fragmentcomprising two linked Fab fragments (vii) single chain Fv molecules(scFv), wherein a VH domain and a VL domain are linked by a peptidelinker which allows the two domains to associate to form an antigenbinding site (Bird et al., (1988) Science, 242, 423-426, Huston et al.,(1988) PNAS USA, 85, 5879-5883); (viii) bispecific single chain Fvdimers (PCT/US92/09965) and (ix) “diabodies”, multivalent ormultispecific fragments constructed by gene fusion (WO94/13804;Holliger, P. (1993) et al., Proc. Natl. Acad. Sci. USA 90 6444-6448,).Fv, scFv or diabody molecules may be stabilized by the incorporation ofdisulphide bridges linking the VH and VL domains (Reiter, Y. et al.,Nature Biotech, 14, 1239-1245, 1996). Minibodies comprising a scFvjoined to a CH3 domain may also be made (Hu, S. et al., (1996) CancerRes., 56, 3055-3061). Other examples of binding fragments are Fab′,which differs from Fab fragments by the addition of a few residues atthe carboxyl terminus of the heavy chain CH1 domain, including one ormore cysteines from the antibody hinge region, and Fab′-SH, which is aFab′ fragment in which the cysteine residue(s) of the constant domainsbear a free thiol group.

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor. Epitopic determinantsusually consist of chemically active surface groupings of molecules suchas amino acids or sugar side chains and may, but not always, havespecific three-dimensional structural characteristics, as well asspecific charge characteristics. An antibody is said to specificallybind an antigen when the dissociation constant is ≦1 μM, preferably ≦100nM and most preferably ≦10 nM.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

“Active” or “activity” in regard to a αVβ6 heterodimeric polypeptiderefers to a portion of an αVβ6 heterodimeric polypeptide that has abiological or an immunological activity of a native αVβ6 polypeptide.“Biological” when used herein refers to a biological function thatresults from the activity of the native αVβ6 polypeptide. A preferredαVβ6 biological activity includes, for example, αVβ6 induced celladhesion.

“Mammal” when used herein refers to any animal that is considered amammal. Preferably, the mammal is human.

Digestion of antibodies with the enzyme, papain, results in twoidentical antigen-binding fragments, known also as “Fab” fragments, anda “Fc” fragment, having no antigen-binding activity but having theability to crystallize. Digestion of antibodies with the enzyme, pepsin,results in the a F(ab′)₂ fragment in which the two arms of the antibodymolecule remain linked and comprise two-antigen binding sites. TheF(ab′)₂ fragment has the ability to crosslink antigen.

“Fv” when used herein refers to the minimum fragment of an antibody thatretains both antigen-recognition and antigen-binding sites.

“Fab” when used herein refers to a fragment of an antibody thatcomprises the constant domain of the light chain and the CH1 domain ofthe heavy chain.

The term “mAb” refers to monoclonal antibody.

“Liposome” when used herein refers to a small vesicle that may be usefulfor delivery of drugs that may include the αVβ6 polypeptide of theinvention or antibodies to such an αVβ6 polypeptide to a mammal.

“Label” or “labeled” as used herein refers to the addition of adetectable moiety to a polypeptide, for example, a radiolabel,fluorescent label, enzymatic label chemiluminescent labeled or abiotinyl group. Radioisotopes or radionuclides may include ³H, ¹⁴C, ¹⁵N,³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, fluorescent labels may includerhodamine, lanthanide phosphors or FITC and enzymatic labels may includehorseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase.

Additional labels include, by way of illustration and not limitation:enzymes, such as glucose-6-phosphate dehydrogenase (“G6PDH”),alpha-D-galactosidase, glucose oxydase, glucose amylase, carbonicanhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase andperoxidase; dyes; additional fluorescent labels or fluorescers include,such as fluorescein and its derivatives, fluorochrome, GFP (GFP for“Green Fluorescent Protein”), dansyl, umbelliferone, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine;fluorophores such as lanthanide cryptates and chelates e.g. Europium etc(Perkin Elmer and Cis Biointernational); chemoluminescent labels orchemiluminescers, such as isoluminol, luminol and the dioxetanes;sensitizers; coenzymes; enzyme substrates; particles, such as latex orcarbon particles; metal sol; crystallite; liposomes; cells, etc., whichmay be further labelled with a dye, catalyst or other detectable group;molecules such as biotin, digoxygenin or 5-bromodeoxyuridine; toxinmoieties, such as for example a toxin moiety selected from a group ofPseudomonas exotoxin (PE or a cytotoxic fragment or mutant thereof),Diptheria toxin or a cytotoxic fragment or mutant thereof, a botulinumtoxin A, B, C, D, E or F, ricin or a cytotoxic fragment thereof e.g.ricin A, abrin or a cytotoxic fragment thereof, saporin or a cytotoxicfragment thereof, pokeweed antiviral toxin or a cytotoxic fragmentthereof and bryodin 1 or a cytotoxic fragment thereof.

The term “pharmaceutical agent or drug” as used herein refers to achemical compound or composition capable of inducing a desiredtherapeutic effect when properly administered to a patient. Otherchemistry terms herein are used according to conventional usage in theart, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)), (incorporatedherein by reference).

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 percent of allmacromolecular species present in the composition, more preferably morethan about 85%, 90%, 95%, and 99%. Most preferably, the object speciesis purified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.

The term “patient” includes human and veterinary subjects.

Human Antibodies and Humanization of Antibodies

Human antibodies avoid some of the problems associated with antibodiesthat possess murine or rat variable and/or constant regions. Thepresence of such murine or rat derived proteins can lead to the rapidclearance of the antibodies or can lead to the generation of an immuneresponse against the antibody by a patient. In order to avoid theutilization of murine or rat derived antibodies, fully human antibodiescan be generated through the introduction of functional human antibodyloci into a rodent, other mammal or animal so that the rodent, othermammal or animal produces fully human antibodies.

One method for generating fully human antibodies is through the use ofXenoMouse® strains of mice that have been engineered to contain up tobut less than 1000 kb-sized germline configured fragments of the humanheavy chain locus and kappa light chain locus. See Mendez et al., NatureGenetics 15:146-156 (1997) and Green and Jakobovits J. Exp. Med.188:483-495 (1998). The XenoMouse® strains are available from Amgen,Inc. (Fremont, Calif.).

The production of the XenoMouse® strains of mice is further discussedand delineated in U.S. patent application Ser. No. 07/466,008, filedJan. 12, 1990, Ser. No. 07/610,515, filed Nov. 8, 1990, Ser. No.07/919,297, filed Jul. 24, 1992, Ser. No. 07/922,649, filed Jul. 30,1992, Ser. No. 08/031,801, filed Mar. 15, 1993, Ser. No. 08/112,848,filed Aug. 27, 1993, Ser. No. 08/234,145, filed Apr. 28, 1994, Ser. No.08/376,279, filed Jan. 20, 1995, Ser. No. 08/430, 938, filed Apr. 27,1995, Ser. No. 08/464,584, filed Jun. 5, 1995, Ser. No. 08/464,582,filed Jun. 5, 1995, Ser. No. 08/463,191, filed Jun. 5, 1995, Ser. No.08/462,837, filed Jun. 5, 1995, Ser. No. 08/486,853, filed Jun. 5, 1995,Ser. No. 08/486,857, filed Jun. 5, 1995, Ser. No. 08/486,859, filed Jun.5, 1995, Ser. No. 08/462,513, filed Jun. 5, 1995, Ser. No. 08/724,752,filed Oct. 2, 1996, Ser. No. 08/759,620, filed Dec. 3, 1996, U.S.Publication 2003/0093820, filed Nov. 30, 2001 and U.S. Pat. Nos.6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598 and JapanesePatent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2. See alsoEuropean Patent No., EP 0 463 151 B1, grant published Jun. 12, 1996,International Patent Application No., WO 94/02602, published Feb. 3,1994, International Patent Application No., WO 96/34096, published Oct.31, 1996, WO 98/24893, published Jun. 11, 1998, WO 00/76310, publishedDec. 21, 2000. The disclosures of each of the above-cited patents,applications, and references are hereby incorporated by reference intheir entirety.

In an alternative approach, others, including GenPharm International,Inc., have utilized a “minilocus” approach. In the minilocus approach,an exogenous Ig locus is mimicked through the inclusion of pieces(individual genes) from the Ig locus. Thus, one or more V_(H) genes, oneor more D_(H) genes, one or more J_(H) genes, a mu constant region, andusually a second constant region (preferably a gamma constant region)are formed into a construct for insertion into an animal. This approachis described in U.S. Pat. No. 5,545,807 to Surani et al., and U.S. Pat.Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458 each toLonberg and Kay, U.S. Pat. Nos. 5,591,669 and 6,023.010 to Krimpenfortand Berns, U.S. Pat. Nos. 5,612,205, 5,721,367, and 5,789,215 to Bernset al., and U.S. Pat. No. 5,643,763 to Choi and Dunn, and GenPharmInternational U.S. patent application Ser. No. 07/574,748, filed Aug.29, 1990, Ser. No. 07/575,962, filed Aug. 31, 1990, Ser. No. 07/810,279,filed Dec. 17, 1991, Ser. No. 07/853,408, filed Mar. 18, 1992, Ser. No.07/904,068, filed Jun. 23, 1992, Ser. No. 07/990,860, filed Dec. 16,1992, Ser. No. 08/053,131, filed Apr. 26, 1993, Ser. No. 08/096,762,filed Jul. 22, 1993, Ser. No. 08/155,301, filed Nov. 18, 1993, Ser. No.08/161,739, filed Dec. 3, 1993, Ser. No. 08/165,699, filed Dec. 10,1993, Ser. No. 08/209,741, filed Mar. 9, 1994, the disclosures of whichare hereby incorporated by reference. See also European Patent No. 0 546073 B1, International Patent Application Nos. WO 92/03918, WO 92/22645,WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO96/14436, WO 97/13852, and WO 98/24884 and U.S. Pat. No. 5,981,175, thedisclosures of which are hereby incorporated by reference in theirentirety. See further Taylor et al., 1992, Chen et al., 1993, Tuaillonet al., 1993, Choi et al., 1993, Lonberg et al., (1994), Taylor et al.,(1994), and Tuaillon et al., (1995), Fishwild et al., (1996), thedisclosures of which are hereby incorporated by reference in theirentirety.

Kirin has also demonstrated the generation of human antibodies from micein which, through microcell fusion, large pieces of chromosomes, orentire chromosomes, have been introduced. See European PatentApplication Nos. 773 288 and 843 961, the disclosures of which arehereby incorporated by reference. Additionally, KM™—mice, which are theresult of cross-breeding of Kirin's Tc mice with Medarex's minilocus(Humab) mice have been generated. These mice possess the human IgHtranschromosome of the Kirin mice and the kappa chain transgene of theGenpharm mice (Ishida et al., Cloning Stem Cells, (2002) 4:91-102).

Human antibodies can also be derived by in vitro methods. Suitableexamples include but are not limited to phage display (CAT, Morphosys,Dyax, Biosite/Medarex, Xoma, Symphogen, Alexion (formerly Proliferon),Affimed) ribosome display (CAT), yeast display, and the like.

Preparation of Antibodies

Antibodies, as described herein, were prepared through the utilizationof the XenoMouse® technology, as described below. Such mice, then, arecapable of producing human immunoglobulin molecules and antibodies andare deficient in the production of murine immunoglobulin molecules andantibodies. Technologies utilized for achieving the same are disclosedin the patents, applications, and references disclosed in the backgroundsection herein. In particular, however, a preferred embodiment oftransgenic production of mice and antibodies therefrom is disclosed inU.S. patent application Ser. No. 08/759,620, filed Dec. 3, 1996 andInternational Patent Application Nos. WO 98/24893, published Jun. 11,1998 and WO 00/76310, published Dec. 21, 2000, the disclosures of whichare hereby incorporated by reference. See also Mendez et al., NatureGenetics 15:146-156 (1997), the disclosure of which is herebyincorporated by reference.

Through the use of such technology, fully human monoclonal antibodies toa variety of antigens have been produced. Essentially, XenoMouse® linesof mice are immunized with an antigen of interest (e.g. αVβ6), lymphaticcells (such as B-cells) are recovered from the hyper-immunized mice, andthe recovered lymphocytes are fused with a myeloid-type cell line toprepare immortal hybridoma cell lines. These hybridoma cell lines arescreened and selected to identify hybridoma cell lines that producedantibodies specific to the antigen of interest. Provided herein aremethods for the production of multiple hybridoma cell lines that produceantibodies specific to αVβ6. Further, provided herein arecharacterization of the antibodies produced by such cell lines,including nucleotide and amino acid sequence analyses of the heavy andlight chains of such antibodies.

Alternatively, instead of being fused to myeloma cells to generatehybridomas, B cells can be directly assayed. For example, CD19+ B cellscan be isolated from hyperimmune XenoMouse® mice and allowed toproliferate and differentiate into antibody-secreting plasma cells.Antibodies from the cell supernatants are then screened by ELISA forreactivity against the αVβ6 immunogen. The supernatants might also bescreened for immunoreactivity against fragments of αVβ6 to further mapthe different antibodies for binding to domains of functional intereston αVβ6. The antibodies may also be screened against other related humanintegrins and against the rat, the mouse, and non-human primate, such asCynomolgus monkey, orthologues of αVβ6, the last to determine speciescross-reactivity. B cells from wells containing antibodies of interestmay be immortalized by various methods including fusion to makehybridomas either from individual or from pooled wells, or by infectionwith EBV or transfection by known immortalizing genes and then platingin suitable medium. Alternatively, single plasma cells secretingantibodies with the desired specificities are then isolated using aαVβ6-specific hemolytic plaque assay (see for example Babcook et al.,Proc. Natl. Acad. Sci. USA 93:7843-48 (1996)). Cells targeted for lysisare preferably sheep red blood cells (SRBCs) coated with the αVβ6antigen.

In the presence of a B-cell culture containing plasma cells secretingthe immunoglobulin of interest and complement, the formation of a plaqueindicates specific αVβ6-mediated lysis of the sheep red blood cellssurrounding the plasma cell of interest. The single antigen-specificplasma cell in the center of the plaque can be isolated and the geneticinformation that encodes the specificity of the antibody is isolatedfrom the single plasma cell. Using reverse-transcription followed by PCR(RT-PCR), the DNA encoding the heavy and light chain variable regions ofthe antibody can be cloned. Such cloned DNA can then be further insertedinto a suitable expression vector, preferably a vector cassette such asa pcDNA, more preferably such a pcDNA vector containing the constantdomains of immunglobulin heavy and light chain. The generated vector canthen be transfected into host cells, e.g., HEK293 cells, CHO cells, andcultured in conventional nutrient media modified as appropriate forinducing transcription, selecting transformants, or amplifying the genesencoding the desired sequences.

In general, antibodies produced by the fused hybridomas were human IgG2heavy chains with fully human kappa or lambda light chains. Antibodiesdescribed herein possess human IgG4 heavy chains as well as IgG2 heavychains. Antibodies can also be of other human isotypes, including IgG1.The antibodies possessed high affinities, typically possessing a Kd offrom about 10⁻⁶ through about 10⁻¹² M or below, when measured by solidphase and solution phase techniques. Antibodies possessing a Kd of atleast 10⁻¹¹ M are preferred to inhibit the activity of αVβ6.

As will be appreciated, antibodies can be expressed in cell lines otherthan hybridoma cell lines. Sequences encoding particular antibodies canbe used to transform a suitable mammalian host cell. Transformation canbe by any known method for introducing polynucleotides into a host cell,including, for example packaging the polynucleotide in a virus (or intoa viral vector) and transducing a host cell with the virus (or vector)or by transfection procedures known in the art, as exemplified by U.S.Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which patentsare hereby incorporated herein by reference). The transformationprocedure used depends upon the host to be transformed. Methods forintroducing heterologous polynucleotides into mammalian cells are wellknown in the art and include dextran-mediated transfection, calciumphosphate precipitation, polybrene mediated transfection, protoplastfusion, electroporation, encapsulation of the polynucleotide(s) inliposomes, and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC), including but not limited toChinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK)cells, monkey kidney cells (COS), human hepatocellular carcinoma cells(e.g., Hep G2), human epithelial kidney 293 cells, and a number of othercell lines. Cell lines of particular preference are selected throughdetermining which cell lines have high expression levels and produceantibodies with constitutive αVβ6 binding properties.

Based on the ability of mAbs to significantly neutralize αVβ6 activity(as demonstrated in the Examples below), these antibodies will havetherapeutic effects in treating symptoms and conditions resulting fromαVβ6 expression. In specific embodiments, the antibodies and methodsherein relate to the treatment of symptoms resulting from αVβ6 inducedcell adhesion or signaling induced as a result of avb6 interaction withit s ligands

According to another aspect of the invention there is provided apharmaceutical composition comprising an antagonist of the biologicalactivity of αVβ6, and a pharmaceutically acceptable carrier. In oneembodiment the antagonist comprises an antibody. According to anotheraspect of the invention there is provided a pharmaceutical compositioncomprising an antagonist of the biological activity of αVβ6, and apharmaceutically acceptable carrier. In one embodiment the antagonistcomprises an antibody.

Anti-αVβ6 antibodies are useful in the detection of αVβ6 in patientsamples and accordingly are useful as diagnostics for disease states asdescribed herein. In addition, based on their ability to significantlyinhibit αVβ6 activity (as demonstrated in the Examples below), anti-αVβ6antibodies have therapeutic effects in treating symptoms and conditionsresulting from αVβ6 expression. In specific embodiments, the antibodiesand methods herein relate to the treatment of symptoms resulting fromαVβ6 induced cell adhesion. Further embodiments involve using theantibodies and methods described herein to treat an αVβ6 related diseaseor disorder including neoplastic diseases, such as, melanoma, small celllung cancer, non-small cell lung cancer, glioma, hepatocellular (liver)carcinoma, thyroid tumor, gastric (stomach) cancer, prostate cancer,breast cancer, ovarian cancer, bladder cancer, lung cancer,glioblastoma, endometrial cancer, kidney cancer, colon cancer, andpancreatic cancer.

Therapeutic Administration and Formulations

Embodiments of the invention include sterile pharmaceutical formulationsof anti-αVβ6 antibodies that are useful as treatments for diseases. Suchformulations would inhibit the binding of ligands to the αVβ6 integrin,thereby effectively treating pathological conditions where, for example,tissue αVβ6 is abnormally elevated. Anti-αVβ6 antibodies preferablypossess adequate affinity to potently inhibit αVβ6 activity, andpreferably have an adequate duration of action to allow for infrequentdosing in humans. A prolonged duration of action will allow for lessfrequent and more convenient dosing schedules by alternate parenteralroutes such as subcutaneous or intramuscular injection.

Sterile formulations can be created, for example, by filtration throughsterile filtration membranes, prior to or following lyophilization andreconstitution of the antibody. The antibody ordinarily will be storedin lyophilized form or in solution. Therapeutic antibody compositionsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having an adapter thatallows retrieval of the formulation, such as a stopper pierceable by ahypodermic injection needle.

The route of antibody administration is in accord with known methods,e.g., injection or infusion by intravenous, intraperitoneal,intracerebral, intramuscular, intraocular, intraarterial, intrathecal,inhalation or intralesional routes, direct injection to a tumor site, orby sustained release systems as noted below. The antibody is preferablyadministered continuously by infusion or by bolus injection.

An effective amount of antibody to be employed therapeutically willdepend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it ispreferred that the therapist titer the dosage and modify the route ofadministration as required to obtain the optimal therapeutic effect.Typically, the clinician will administer antibody until a dosage isreached that achieves the desired effect. The progress of this therapyis easily monitored by conventional assays or by the assays describedherein.

Antibodies, as described herein, can be prepared in a mixture with apharmaceutically acceptable carrier. This therapeutic composition can beadministered intravenously or through the nose or lung, preferably as aliquid or powder aerosol (lyophilized). The composition may also beadministered parenterally or subcutaneously as desired. Whenadministered systemically, the therapeutic composition should besterile, pyrogen-free and in a parenterally acceptable solution havingdue regard for pH, isotonicity, and stability. These conditions areknown to those skilled in the art. Briefly, dosage formulations of thecompounds described herein are prepared for storage or administration bymixing the compound having the desired degree of purity withpharmaceutically acceptable carriers, excipients, or stabilizers. Suchmaterials are non-toxic to the recipients at the dosages andconcentrations employed, and include buffers such as TRIS HCl,phosphate, citrate, acetate and other organic acid salts; antioxidantssuch as ascorbic acid; low molecular weight (less than about tenresidues) peptides such as polyarginine, proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidinone; amino acids such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium and/or nonionicsurfactants such as TWEEN, PLURONICS or polyethyleneglycol.

Sterile compositions for injection can be formulated according toconventional pharmaceutical practice as described in Remington: TheScience and Practice of Pharmacy (20^(th) ed, Lippincott Williams &Wilkens Publishers (2003)). For example, dissolution or suspension ofthe active compound in a pharmaceutically acceptable carrier such aswater or naturally occurring vegetable oil like sesame, peanut, orcottonseed oil or a synthetic fatty vehicle like ethyl oleate or thelike may be desired. Buffers, preservatives, antioxidants and the likecan be incorporated according to accepted pharmaceutical practice.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing thepolypeptide, which matrices are in the form of shaped articles, films ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed Mater. Res., (1981) 15:167-277 andLanger, Chem. Tech., (1982) 12:98-105, or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,(1983) 22:547-556), non-degradable ethylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as theLUPRON Depot™ (injectable microspheres composed of lactic acid-glycolicacid copolymer and Ieuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid (EP 133,988).

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated proteinsremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for protein stabilization depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation through disulfideinterchange, stabilization may be achieved by modifying sulfhydrylresidues, lyophilizing from acidic solutions, controlling moisturecontent, using appropriate additives, and developing specific polymermatrix compositions.

The antibodies of the invention also encompass antibodies that havehalf-lives (e.g., serum half-lives) in a mammal, preferably a human, ofgreater than that of an unmodified antibody. In one embodiment, saidantibody anybody half life is greater than days, greater than 20 days,greater than 25 days, greater than 30 days, greater than 35 days,greater than 40 days, greater than 45 days, greater than 2 months,greater than 3 months, greater than 4 months, or greater than 5 months.The increased half-lives of the antibodies of the present invention orfragments thereof in a mammal, preferably a human, result in a higherserum titer of said antibodies or antibody fragments in the mammal, andthus, reduce the frequency of the administration of said antibodies orantibody fragments and/or reduces the concentration of said antibodiesor antibody fragments to be administered. Antibodies or fragmentsthereof having increased in vivo half-lives can be generated bytechniques known to those of skill in the art. For example, antibodiesor fragments thereof with increased in vivo half-lives can be generatedby modifying (e.g., substituting, deleting or adding) amino acidresidues identified as involved in the interaction between the Fc domainand the FcRn receptor (see, e.g., International Publication Nos. WO97/34631 and WO 02/060919, which are incorporated herein by reference intheir entireties). Antibodies or fragments thereof with increased invivo half-lives can be generated by attaching to said antibodies orantibody fragments polymer molecules such as high molecular weightpolyethyleneglycol (PEG). PEG can be attached to said antibodies orantibody fragments with or without a multifunctional linker eitherthrough site-specific conjugation of the PEG to the N- or C-terminus ofsaid antibodies or antibody fragments or via epsilon-amino groupspresent on lysine residues. Linear or branched polymer derivatizationthat results in minimal loss of biological activity will be used. Thedegree of conjugation will be closely monitored by SDS-PAGE and massspectrometry to ensure proper conjugation of PEG molecules to theantibodies. Unreacted PEG can be separated from antibody-PEG conjugatesby, e.g., size exclusion or ion-exchange chromatography.

Sustained-released compositions also include preparations of crystals ofthe antibody suspended in suitable formulations capable of maintainingcrystals in suspension. These preparations when injected subcutaneouslyor intraperitonealy can produce a sustained release effect. Othercompositions also include liposomally entrapped antibodies. Liposomescontaining such antibodies are prepared by methods known per se: U.S.Pat. No. DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA,(1985) 82:3688-3692; Hwang et al., Proc. Natl. Acad. Sci. USA, (1980)77:4030-4034; EP 52,322; EP 36,676; EP 88,046; EP 143,949; 142,641;Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324.

The dosage of the antibody formulation for a given patient will bedetermined by the attending physician taking into consideration variousfactors known to modify the action of drugs including severity and typeof disease, body weight, sex, diet, time and route of administration,other medications and other relevant clinical factors. Therapeuticallyeffective dosages may be determined by either in vitro or in vivomethods.

An effective amount of the antibodies, described herein, to be employedtherapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, and the condition of thepatient. Accordingly, it is preferred for the therapist to titer thedosage and modify the route of administration as required to obtain theoptimal therapeutic effect. A typical daily dosage might range fromabout 0.001 mg/kg to up to 100 mg/kg or more, depending on the factorsmentioned above. Typically, the clinician will administer thetherapeutic antibody until a dosage is reached that achieves the desiredeffect. The progress of this therapy is easily monitored by conventionalassays or as described herein.

It will be appreciated that administration of therapeutic entities inaccordance with the compositions and methods herein will be administeredwith suitable carriers, excipients, and other agents that areincorporated into formulations to provide improved transfer, delivery,tolerance, and the like. These formulations include, for example,powders, pastes, ointments, jellies, waxes, oils, lipids, lipid(cationic or anionic) containing vesicles (such as Lipofectin™), DNAconjugates, anhydrous absorption pastes, oil-in-water and water-in-oilemulsions, emulsions carbowax (polyethylene glycols of various molecularweights), semi-solid gels, and semi-solid mixtures containing carbowax.Any of the foregoing mixtures may be appropriate in treatments andtherapies in accordance with the present invention, provided that theactive ingredient in the formulation is not inactivated by theformulation and the formulation is physiologically compatible andtolerable with the route of administration. See also Baldrick P.“Pharmaceutical excipient development: the need for preclinicalguidance.” Regul. Toxicol. Pharmacol. 32(2):210-8 (2000), Wang W.“Lyophilization and development of solid protein pharmaceuticals.” Int.J. Pharm. 203(1-2):1-60 (2000), Charman W N “Lipids, lipophilic drugs,and oral drug delivery-some emerging concepts.” J. Pharm Sci.89(8):967-78 (2000), Powell et al., “Compendium of excipients forparenteral formulations” PDA J Pharm Sci Technol. 52:238-311 (1998) andthe citations therein for additional information related toformulations, excipients and carriers well known to pharmaceuticalchemists.

Design and Generation of Other Therapeutics

In accordance with the present invention and based on the activity ofthe antibodies that are produced and characterized herein with respectto αVβ6, the design of other therapeutic modalities beyond antibodymoieties is facilitated. Such modalities include, without limitation,advanced antibody therapeutics, such as bispecific antibodies,immunotoxins, and radiolabeled therapeutics, single domain antibodies,generation of peptide therapeutics, αVβ6 binding domains in novelscaffolds, gene therapies, particularly intrabodies, antisensetherapeutics, and small molecules.

In connection with the generation of advanced antibody therapeutics,where complement fixation is a desirable attribute, it may be possibleto sidestep the dependence on complement for cell killing through theuse of bispecific antibodies, immunotoxins, or radiolabels, for example.

Bispecific antibodies can be generated that comprise (i) two antibodiesone with a specificity to αVβ6 and another to a second molecule that areconjugated together, (ii) a single antibody that has one chain specificto αVβ6 and a second chain specific to a second molecule, or (iii) asingle chain antibody that has specificity to αVβ6 and the othermolecule. Such bispecific antibodies can be generated using techniquesthat are well known; for example, in connection with (i) and (ii) seee.g., Fanger et al., Immunol Methods 4:72-81 (1994) and Wright andHarris, supra. and in connection with (iii) see e.g., Traunecker et al.,Int. J. Cancer (Suppl.) 7:51-52 (1992). In each case, the secondspecificity can be made to the heavy chain activation receptors,including, without limitation, CD16 or CD64 (see e.g., Deo et al.,Immunol. Today 18:127 (1997)) or CD89 (see e.g., Valerius et al., Blood90:4485-4492 (1997)).

In connection with immunotoxins, antibodies can be modified to act asimmunotoxins utilizing techniques that are well known in the art. Seee.g., Vitetta Immunol Today 14:252 (1993). See also U.S. Pat. No.5,194,594. In connection with the preparation of radiolabeledantibodies, such modified antibodies can also be readily preparedutilizing techniques that are well known in the art. See e.g., Junghanset al., in Cancer Chemotherapy and Biotherapy 655-686 (2d edition,Chafner and Longo, eds., Lippincott Raven (1996)). See also U.S. Pat.Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (RE 35,500), 5,648,471,and 5,697,902.

An antigen binding site may be provided by means of arrangement of CDRson non-antibody protein scaffolds, such as fibronectin or cytochrome Betc. (Haan & Maggos (2004) BioCentury, 12(5): A1-A6; Koide et al.,(1998) Journal of Molecular Biology, 284: 1141-1151; Nygren et al.,(1997) Current Opinion in Structural Biology, 7: 463-469) or byrandomising or mutating amino acid residues of a loop within a proteinscaffold to confer binding specificity for a desired target. Scaffoldsfor engineering novel binding sites in proteins have been reviewed indetail by Nygren et al., (Nygren et al., (1997) Current Opinion inStructural Biology, 7: 463-469). Protein scaffolds for antibody mimicsare disclosed in WO/0034784, which is herein incorporated by referencein its entirety, in which the inventors describe proteins (antibodymimics) that include a fibronectin type III domain having at least onerandomised loop. A suitable scaffold into which to graft one or moreCDRs, e.g. a set of HCDRs, may be provided by any domain member of theimmunoglobulin gene superfamily. The scaffold may be a human ornon-human protein. An advantage of a non-antibody protein scaffold isthat it may provide an antigen-binding site in a scaffold molecule thatis smaller and/or easier to manufacture than at least some antibodymolecules. Small size of a binding agent may confer useful physiologicalproperties, such as an ability to enter cells, penetrate deep intotissues or reach targets within other structures, or to bind withinprotein cavities of the target antigen. Use of antigen binding sites innon-antibody protein scaffolds is reviewed in Wess, 2004 (Wess, L. In:BioCentury, The Bernstein Report on BioBusiness, 12(42), A1-A7, 2004).Typical are proteins having a stable backbone and one or more variableloops, in which the amino acid sequence of the loop or loops isspecifically or randomly mutated to create an antigen-binding site thatbinds the target antigen. Such proteins include the IgG-binding domainsof protein A from S. aureus, transferrin, albumin, tetranectin,fibronectin (e.g. 10th fibronectin type III domain), lipocalins as wellas gamma-crystalline and other Affilin™ scaffolds (Scil Proteins).Examples of other approaches include synthetic “Microbodies” based oncyclotides—small proteins having intra-molecular disulphide bonds,Microproteins (Versabodies™, Amunix) and ankyrin repeat proteins(DARPins, Molecular Partners).

In addition to antibody sequences and/or an antigen-binding site, abinding agent according to the present invention may comprise otheramino acids, e.g. forming a peptide or polypeptide, such as a foldeddomain, or to impart to the molecule another functional characteristicin addition to ability to bind antigen. Binding agents of the inventionmay carry a detectable label, or may be conjugated to a toxin or atargeting moiety or enzyme (e.g. via a peptidyl bond or linker). Forexample, a binding agent may comprise a catalytic site (e.g. in anenzyme domain) as well as an antigen binding site, wherein the antigenbinding site binds to the antigen and thus targets the catalytic site tothe antigen. The catalytic site may inhibit biological function of theantigen, e.g. by cleavage.

Combinations

The anti-tumor treatment defined herein may be applied as a sole therapyor may involve, in addition to the compounds of the invention,conventional surgery or radiotherapy or chemotherapy. Such chemotherapymay include one or more of the following categories of anti tumoragents:

(i) other antiproliferative/antineoplastic drugs and combinationsthereof, as used in medical oncology, such as alkylating agents (forexample cis-platin, oxaliplatin, carboplatin, cyclophosphamide, nitrogenmustard, melphalan, chlorambucil, busulphan, temozolamide andnitrosoureas); antimetabolites (for example gemcitabine and antifolatessuch as fluoropyrimidines like 5-fluorouracil and tegafur, raltitrexed,methotrexate, cytosine arabinoside, and hydroxyurea); antitumourantibiotics (for example anthracyclines like adriamycin, bleomycin,doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,dactinomycin and mithramycin); antimitotic agents (for example vincaalkaloids like vincristine, vinblastine, vindesine and vinorelbine andtaxoids like taxol and taxotere and polokinase inhibitors); andtopoisomerase inhibitors (for example epipodophyllotoxins like etoposideand teniposide, amsacrine, topotecan and camptothecin);

(ii) cytostatic agents such as antioestrogens (for example tamoxifen,fulvestrant, toremifene, raloxifene, droloxifene and iodoxyfene),antiandrogens (for example bicalutamide, flutamide, nilutamide andcyproterone acetate), LHRH antagonists or LHRH agonists (for examplegoserelin, leuprorelin and buserelin), progestogens (for examplemegestrol acetate), aromatase inhibitors (for example as anastrozole,letrozole, vorazole and exemestane) and inhibitors of 5α-reductase suchas finasteride;

(iii) anti-invasion agents (for example c-Src kinase family inhibitorslike4-(6-chloro-2,3-methylenedioxyanilino)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-tetrahydropyran-4-yloxyquinazoline(AZD0530; International Patent Application WO 01/94341) andN-(2-chloro-6-methylphenyl)-2-{6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-methylpyrimidin-4-ylamino}thiazole-5-carboxamide(dasatinib, BMS-354825; J. Med. Chem., 2004, 47, 6658-6661), andmetalloproteinase inhibitors like marimastat, inhibitors of urokinaseplasminogen activator receptor function or antibodies to Heparanase);

(iv) inhibitors of growth factor function: for example such inhibitorsinclude growth factor antibodies and growth factor receptor antibodies(for example the anti-erbB2 antibody trastuzumab [Herceptin™], theanti-EGFR antibody panitumumab, the anti-EGFR inhibitor Bevacizumab(Avastin™), the anti-erbB1 antibody cetuximab [Erbitux, C225] and anygrowth factor or growth factor receptor antibodies disclosed by Stem etal., Critical reviews in oncology/haematology, 2005, Vol. 54, pp11-29);such inhibitors also include tyrosine kinase inhibitors, for exampleinhibitors of the epidermal growth factor family (for example EGFRfamily tyrosine kinase inhibitors such asN-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine(gefitinib, ZD1839),N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine(erlotinib, OSI-774) and6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)-quinazolin-4-amine(CI 1033), erbB2 tyrosine kinase inhibitors such as lapatinib,inhibitors of the hepatocyte growth factor family, inhibitors of theplatelet-derived growth factor family such as imatinib, inhibitors ofserine/threonine kinases (for example Ras/Raf signalling inhibitors suchas farnesyl transferase inhibitors, for example sorafenib (BAY43-9006)), inhibitors of cell signalling through MEK and/or AKT kinases,inhibitors of the hepatocyte growth factor family, c-kit inhibitors, ablkinase inhibitors, IGF receptor (insulin-like growth factor) kinaseinhibitors; aurora kinase inhibitors (for example AZD1152, PH739358,VX-680, MLN8054, R763, MP235, MP529, VX-528 AND AX39459) and cyclindependent kinase inhibitors such as CDK2 and/or CDK4 inhibitors;

(v) antiangiogenic agents such as those which inhibit the effects ofvascular endothelial growth factor, [for example the anti-vascularendothelial cell growth factor antibody bevacizumab (Avastin™) and VEGFreceptor tyrosine kinase inhibitors such as4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline(ZD6474; Example 2 within WO 01/32651),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline(AZD2171; Example 240 within WO 00/47212), vatalanib (PTK787; WO98/35985) and SU11248 (sunitinib; WO 01/60814), compounds such as thosedisclosed in International Patent Applications WO97/22596, WO 97/30035,WO 97/32856 and WO 98/13354 and compounds that work by other mechanisms(for example linomide, inhibitors of integrin αvβ3 function andangiostatin);

(vi) vascular damaging agents such as Combretastatin A4 and compoundsdisclosed in International Patent Applications WO 99/02166, WO 00/40529,WO 00/41669, WO 01/92224, WO 02/04434 and WO 02/08213;

(vii) antisense therapies, for example those which are directed to thetargets listed above, such as ISIS 2503, an anti-ras antisense;

(viii) gene therapy approaches, including for example approaches toreplace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2,GDEPT (gene-directed enzyme pro-drug therapy) approaches such as thoseusing cytosine deaminase, thymidine kinase or a bacterial nitroreductaseenzyme and approaches to increase patient tolerance to chemotherapy orradiotherapy such as multi-drug resistance gene therapy; and

(ix) immunotherapy approaches, including for example ex-vivo and in-vivoapproaches to increase the immunogenicity of patient tumour cells, suchas transfection with cytokines such as interleukin 2, interleukin 4 orgranulocyte-macrophage colony stimulating factor, approaches to decreaseT-cell anergy, approaches using transfected immune cells such ascytokine-transfected dendritic cells, approaches usingcytokine-transfected tumor cell lines and approaches usinganti-idiotypic antibodies.

Such conjoint treatment may be achieved by way of the simultaneous,sequential or separate dosing of the individual components of thetreatment. Such combination products employ the compounds of thisinvention, or pharmaceutically acceptable salts thereof, within thedosage range described hereinbefore and the other pharmaceuticallyactive agent within its approved dosage range.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting upon the teachings herein.

Example 1 Immunization and Titering

Immunization

Immunizations were conducted using soluble αVβ6 and cell-bound αVβ6 (CHOtransfectants expressing human αVβ6 at the cell surface), respectively.For the generation of CHO transfectants, human full length αVβ6 cDNA wasinserted into the pcDNA 3 expression vector. CHO cells were transientlytransfected via electroporation. Expression of human αVβ6 on the cellsurface at the level suitable for immunogen purpose was confirmed byFluorescene-Activated Cell Sorter (FACS) analysis. Ten fig/mouse ofsoluble protein for Campaign 1, and 3×10⁶ cells/mouse of transfected CHOcells for Campaign 2, were used for initial immunization in XenoMouse™according to the methods disclosed in U.S. patent application Ser. No.08/759,620, filed Dec. 3, 1996 and International Patent Application Nos.WO 98/24893, published Jun. 11, 1998 and WO 00/76310, published Dec. 21,2000, the disclosures of which are hereby incorporated by reference.Following the initial immunization, thirteen subsequent boostimmunizations of five μg/mouse were administered for groups one and two(soluble antigen), and nine subsequent boost immunizations of 1.5×10⁶cells/mouse were administered for groups three and four (cell-boundantigen). The immunization programs are summarized in Table 2.

TABLE 2 Summary of Immunization Programs No of Immunization CampaignGroup Immunogen Strain mice routes 1 1 Soluble αVβ6 XMG2/k 10 IP, Tail,BIP, twice/wk, x 6wks 1 2 Soluble αVβ6 XMG1/kl 10 IP, Tail, BIP,twice/wk, x 6wks 2 3 Cell-bound αVβ6 XMG2/k 10 IP, Tail, BIP, (CHOtwice/wk, x transfectants) 6wks 2 4 Cell-bound αVβ6 XMG1/kl 10 IP, Tail,BIP, (CHO twice/wk, x transfectants) 6wksSelection of Animals for Harvest by Titer

Titers of the antibody against human αVβ6 were tested by ELISA assay formice immunized with soluble antigen. Titers of the antibody for miceimmunized with native (cell-bound) antigen were tested by FACS. TheELISA and FACS analyses showed that there were some mice which appearedto be specific for αVβ6. Therefore, at the end of the immunizationprogram, twenty mice were selected for harvest, and lymphocytes wereisolated from the spleens and lymph nodes of the immunized mice, asdescribed in Example 2.

Example 2 Recovery of Lymphocytes and B-Cell Isolations

Immunized mice were sacrificed by cervical dislocation, and the draininglymph nodes harvested and pooled from each cohort. The lymphoid cellswere dissociated by grinding in DMEM to release the cells from thetissues and the cells were suspended in DMEM. B cells were enriched bynegative selection in IgM and positive selection on IgG. The cells werecultured to allow B cell expansion and differentiation intoantibody-secreting plasma cells.

Antibody-secreting plasma cells were grown as routine in the selectivemedium. Exhaustive supernatants collected from the cells thatpotentially produce anti-human αVβ6 antibodies were subjected tosubsequent screening assays as detailed in the examples below.

Example 3 Binding to Cell-Bound αVβ6

The binding of secreted antibodies to αVβ6 was assessed. Binding tocell-bound αVβ6 was assessed using an FMAT macroconfocal scanner, andbinding to soluble αVβ6 was analyzed by ELISA, as described below.

Supernatants collected from harvested cells were tested to assess thebinding of secreted antibodies to HEK 293 cells stably overexpressingαVβ6. A parental 293F cell line was used as a negative control. Cells inFreestyle media (Invitrogen) were seeded into 384-well FMAT plates in avolume of 50 μL/well at a density of 2500 cells/well for the stabletransfectants, and at a density of 22,500 cells/well for the parentalcells, and cells were incubated overnight at 37° C. Then, 10 μL/well ofsupernatant was added, and plates were incubated for approximately onehour at 4° C., after which 10 μL/well of anti-human IgG-Cy5 secondaryantibody was added at a concentration of 2.8 μg/ml (400 ng/ml finalconcentration). Plates were then incubated for one hour at 4° C., andfluorescence was read using an FMAT macroconfocal scanner (AppliedBiosystems). FMAT results for 11 antibodies are summarized in Table 3.

Additionally, antibody binding to soluble αVβ6 was analyzed by ELISA.Costar medium binding 96-well plates (Costar catalog #3368) were coatedby incubating overnight at 4° C. with αVβ6 at a concentration of 5 μg/mlin TBS/1 mM MgCl₂ buffer for a total volume of 50 μL/well. Plates werethen washed with TBS/1 mM MgCl₂ buffer, and blocked with 250 μL of1×PBS/1% milk for thirty minutes at room temperature. Ten μL ofsupernatant was then added to 40 μL TBS/1 mM MgCl₂/1% milk and incubatedfor one hour at room temperature. Plates were washed and then incubatedwith goat-anti-human IgG Fc-peroxidase at 0.400 ng/ml in TBS/1 mMMgCl₂/1% milk, and incubated for one hour at room temperature. Plateswere washed and then developed with 1-Step TMB substrate. The ELISAresults for one of the antibodies are shown in Table 3.

TABLE 3 Binding of Supernatants to Cell-Bound and Soluble αVβ6 ELISAFMAT Data data mAb Count FL1 FL1XCount OD sc 049 185 4377.73 809880 NDsc 058 ND ND ND 1.79 sc 188 127 628.04 79761 ND sc 097 98 1237.18 121243ND sc 277 28 382.31 10704 ND sc 133 82 709.82 58205 ND sc 161 23 725.2116679 ND sc 254 174 9179.65 1597259 ND sc 264 63 734.29 46260 ND sc 298102 2137.94 218069 ND sc 374 174 4549.65 791639 ND sc 320 141 3014.63425062 ND Negative Control (Blank): 0 0 0 0.21 Positive Control (2077z-1ug/mL): 67 659.49 44185 6.00

Example 4 Inhibition of Cell Adhesion

In order to determine the relative potency of the differentantibody-containing supernatants, the antibodies were assessed for theirability to inhibit TGFβLAP-mediated adhesion of αVβ6-positive HT29cells. Plates were coated overnight with 10 g/ml TGFβLAP, andpre-blocked with 3% BSA/PBS for 1 hour prior to the assay. Cells werethen pelleted and washed twice in HBBS, after which the cells were thenresuspended in HBSS at appropriate concentrations. The cells wereincubated in the presence of appropriate antibodies at 4° C. for 30minutes in a V-bottom plate. The antigen coating solution was removedand the plates were blocked with 100 μL of 3% BSA for one hour at roomtemperature. Plates were washed twice with PBS or HBSS, and thecell-antibody mixtures were transferred to the coated plate and theplate was incubated at 37° C. for 30 minutes. The cells on the coatedplates were then washed four times in warm HBSS, and the cells werethereafter frozen at −80° C. for one hour. The cells were allowed tothaw at room temperature for one hour, and then 100 μL of CyQuantdye/lysis buffer (Molecular Probes) was added to each well according tothe manufacturer's instructions. Fluorescence was read at an excitationwavelength of 485 nm and an emission wavelength of 530 nm. The resultsfor twelve antibodies are summarized in Table 4. Those antibodies shownranged in potency from 62% inhibition to over 100% inhibition, relativeto coated and uncoated control wells on the plate which were used torepresent the maximum and minimum adhesion values that could be obtainedin the assay.

TABLE 4 Adhesion Assay Antibody Assay 1% Assay 2% Average ID InhibitionInhibition Inhibition sc 049  80%  98%  89% sc 058  77%  46%  62% sc 097 96% 106% 101% sc 133  99% 106% 103% sc 161  98% 106% 102% sc 188  99%103% 101% sc 254  98% 106% 102% sc 264  98% 100%  99% sc 277  98% 101%100% sc 298  98% 102% 100% sc 320  97%  97%  97% sc 374 118%  89% 104%

Example 5 Cross-Reactivity to Macaque αVβ6 and Human αV

Cross-reactivity of the antibody-containing supernatants to macaque αVβ6was tested on the supernatants using FACS analysis on HEK-293 cellstransiently transfected with cynomolgus αV and cynomolgus β6.

Cross-reactivity to human αV was also tested. For this assay,cross-reactivity was tested on the supernatants using FACS analysis onparental A375M cells, which express αVβ3 and αVβ5, but not αVβ6. Thisscreen was designed to show that the antibodies were specificallyrecognizing either the β6 chain or the β6 chain in combination with αV.The human αV assay was run at the same time as the macaque αVβ6cross-reactivity screen.

The assays were performed as follows. A375M cells that wereapproximately 75% confluent were labeled with CFSE intracellular dye bydissociating and then pelleting cells (equivalent to 250,000 to 300,000cells per well) in a falcon tube, then resuspending in 0.125 μM CFSE inFACS buffer to a final volume of 100 μL for every 250,000 cells, andthen by incubating at 37° C. for five minutes. The cells were thenpelleted, the supernatant discarded, and resuspended in FACS buffer andincubated for 30 minutes at 37° C. Cells were then washed twice withFACS buffer and resuspended in a final volume of 100 μL FACS buffer perwell.

HEK-293 cells were transiently transfected with cynomolgus αV andcynomolgus β6. After 48 hours, the cells were collected and resuspendedin FACS buffer to reach a final concentration of approximately 50,000cells in 100 μL.

Approximately 100,000 cells total, comprising a 50:50 mix ofCFSE-labeled A375M cells and transfected 293 cells, were used in eachreaction as follows. 100 μL of CFSE-labeled A375M cells and 100 μL of293 cells were dispensed into a V-bottom plate. The cells in the platewere pelleted at 1500 rpm for 3 minutes, and then resuspended in 100 μLFACS buffer. The pelleting step was repeated, and the FACS buffersupernatant was removed. The harvested antibody-containing supernatants,or control primary antibodies were added in a volume of 50 μL and thecells were resuspended. Primary antibody controls were murine αVβ6(Cat#MAB2077z, Chemicon) and an anti-αV recombinant. The plate wasincubated on ice for 45 minutes, after which 100 μL FACS buffer wasadded to dilute the primary antibody. The cells were then pelleted bycentrifuging at 1500 rpm for 3 minutes, and the pellet was resuspendedin 100 μL FACS buffer. The pelleting step was repeated, and the FACSbuffer supernatant was removed. Cells were then resuspended in theappropriate secondary antibody (5 μg/ml) with 7AAD dye (10 μg/ml), andstained on ice for 7 minutes. Then 150 μL of FACS buffer was added andthe cells were pelLeted at 1500 rpm for 3 minutes, after which the cellswere washed in 100 μL FACS buffer, pelleted, and then resuspended in 250μL buffer and added to FACS tubes. Samples were analyzed on a highthroughput FACS machine and analyzed using Cell Quest Pro software.

The results for twelve antibodies are summarized in Table 5, anddemonstrate that the antibodies shown were able to specifically bind tomacaque αVβ6 but were not able to non-specifically bind human αV on theparental A375M cells.

TABLE 5 Cross-Reactivity to Macaque αVβ6 and Human αV Mac αVβ6 % CellsMac AVB6 A375M % A375M Antibody ID Shifted GeoMean Cells Shifted GeoMeansc 049 23% 30.19 20% 1.74 sc 058 25% 22.77 18% 1.78 sc 097 35% 37.04 24%1.84 sc 133 32% 35.22 24% 1.79 sc 161 14% 32.98 11% 16.68 sc 188 18%23.9 13% 1.65 sc 254 59% 78.49 55% 2.31 sc 264 55% 66.38 46% 2.35 sc 27735% 33.35 23% 1.86 sc 298 53% 63.08 45% 2.14 sc 320 19% 33.45 15% 23.18sc 374 51% 61.79 39% 2.14 Human IgG 1% (day 1) 9.54 (day 1) 5% (day 1)1.66 (day 1) Isotype 0% (day 2) 7.39 (day 2) 1% (day 2) 7.23 (day 2)Control Mouse IgG2 1% (day 1) 8.85 (day 1) 4% (day 1) 1.67 (day 1) withMurine 0% (day 2) 11.21 (day 2) 3% (day 2) 11.16 (day 2) SecondaryAntibody Positive 42% (day 1) 55.52 (day 1) 30% (day 1) 2.03 (day 1)Control 11% (day 2) 28.11 (day 2) 5% (day 2) 15.36 (day 2) 2077z(1ug/ml)

Example 6 αVβ6-Specific Hemolytic Plaque Assay

Antibody-secreting plasma cells were selected from each harvest for theproduction of recombinant antibodies. Here, a fluorescent plaque assaywas used to identify single plasma cells expressing antibodies againstαVβ6. Then, the single cells were subjected to reverse transcription andpolymerase chain reaction to rescue and amplify the variable heavy andvariable light chains that encoded the initial antibody specificity, asdescribed in Example 7. The preparation of a number of specializedreagents and materials needed to conduct the αVβ6-specific hemolyticplaque assay are described below.

Biotinylation of Sheep Red Blood Cells (SRBC).

SRBC were stored in RPMI media as a 25% stock. A 250 μl SRBC packed-cellpellet was obtained by aliquoting 1.0 mL of the stock into a 15-mLfalcon tube, spinning down the cells and removing the supernatant. Thecell pellet was then re-suspended in 4.75 mL PBS at pH 8.6 in a 50 mLtube. In a separate 50 mL tube, 2.5 mg of Sulfo-NHS biotin was added to45 mL of PBS at pH 8.6. Once the biotin had completely dissolved, 5 mLof SRBCs was added and the tube was rotated at room temperature for 1hour. The SRBCs were centrifuged at 3000 g for 5 minutes. Thesupernatant was drawn off and 25 mL PBS at pH 7.4 was added as a wash.The wash cycle was repeated 3 times, then 4.75 mL immune cell media(RPMI 1640 with 10% FCS) was added to the 250 μl biotinylated-SRBC(B-SRBC) pellet to gently re-suspend the B-SRBC (5% B-SRBC stock). Thestock was stored at 40° C. until needed.

Streptavidin (SA) Coating of B-SRBC.

One mL of the 5% B-SRBC stock was transferred into to a fresh eppendorftube. The B-SRBC cells were pelleted with a pulse spin at 8000 rpm (6800rcf) in a microfuge. The supernatant was then drawn off, the pellet wasre-suspended in 1.0 mL PBS at pH 7.4, and the centrifugation wasrepeated. The wash cycle was repeated 2 times, then the B-SRBC pelletwas resuspended in 1.0 mL of PBS at pH 7.4 to give a final concentrationof 5% (v/v). 10 μl of a 10 mg/mL Streptavidin (CalBiochem, San Diego,Calif.) stock solution was added. The tube was mixed and rotated at RTfor 20 minutes. The washing steps were repeated and the SA-SRBC werere-suspended in 1 mL PBS pH 7.4 (5% (v/v)).

Human αVβ6 Coating of SA-SRBC.

Soluble antigen (lacking the transmembrane domain) was used for coatingthe SRBC. Both chains were used because αVβ6 is only presented on thecell surface as a dimer. The SA-SRBC were coated with thebiotinylated-αVβ6 at 50 μg/mL, mixed and rotated at room temperature for20 minutes. The SRBC were washed twice with 1.0 mL of PBS at pH 7.4 asabove. The Ag-coated SRBC were re-suspended in RPMI (+10% FCS) to afinal concentration of 5% (v/v).

Determination of the Quality of αV/36-SRBC by Immunofluorescence (IF).

10 μl of 5% SA-SRBC and 10 μl of 5% Ag-coated SRBC were each added toseparate fresh 1.5 mL eppendorf tube containing 40 μl of PBS. Humananti-αVβ6 antibodies were added to each sample of SRBCs at 50 μg/mL. Thetubes were rotated at room temperature for 25 min, and the cells werethen washed three times with 100 μl of PBS. The cells were re-suspendedin 50 μl of PBS and incubated with 2 μg/mL Gt-anti Human IgG Fc antibodyconjugated to the photostable fluorescent dye Alexa488 (MolecularProbes, Eugene, Oreg.). The tubes were rotated at room temperature for25 min, followed by washing with 100 μl PBS and re-suspension in 10 μlPBS. 10 μl of the stained cells were spotted onto a clean glassmicroscope slide, covered with a glass coverslip, observed underfluorescent light, and scored on an arbitrary scale of 0-4 to assess thequality of the isolated cells.

Preparation of Plasma Cells.

The contents of a single B cell culture well previously identified asneutralizing for αVβ6 activity (therefore containing a B cell clonesecreting the immunoglobulin of interest), was harvested. The B cellculture present in the well was recovered by addition of RPMI+10% FCS at37° C. The cells were re-suspended by pipetting and then transferred toa fresh 1.5 mL eppendorf tube (final volume approximately 500-700 μl).The cells were centrifuged in a microfuge at 1500 rpm (240 rcf) for 2minutes at room temperature, then the tube was rotated 180 degrees andcentrifuged again for 2 minutes at 1500 rpm. The freeze media was drawnoff and the immune cells were resuspended in 100 μl RPMI (10% FCS), thencentrifuged. This washing with RPMI (10% FCS) was repeated and the cellsre-suspended in 60 μl RPMI (FCS) and stored on ice until ready to use.

Performance of the Hemolytic Plaque Assay.

To the 60 μl sample of immune cells was added 60 μl each of αVβ6-coatedSRBC (5% v/v stock), 4× guinea pig complement (Sigma, Oakville, ON)stock prepared in RPMI (FCS), and 4× enhancing sera stock (1:900 in RPMI(FCS)). The mixture (3-5 μl) was spotted onto plastic lids from 100 mmFalcon tissue culture plates and the spots were covered with undilutedparaffin oil. The slides were incubated at 37° C. for a minimum of 45minutes.

Analysis of Plaque Assay Results.

The coating of the sheep red blood cells with the catalytic domain ofhuman αVβ6 was successful. These Ag-coated red blood cells were thenused to identify antigen-specific plasma cells from the wells shownbelow in Table 6. These cells were then isolated by micromanipulation.After micromanipulation to rescue the antigen-specific plasma cells, thegenes encoding the variable region genes were rescued by RT-PCR on asingle plasma cell, as described further in Example 7.

TABLE 6 Plaque Assay Results Parent Plate ID Plaque Assay Plate RowColumn Assay Single Cells 68 B 10 Fluorescent 45-57 296 D 10 Fluorescent58-59 318 F 1 Hemolytic 60-62 612 G 1 Fluorescent 187-189 752 D 12Fluorescent  95-100 762 D 8 Fluorescent 277-286 766 B 5 Fluorescent132-143, 147-150 827 E 12 Fluorescent 159-170 659 F 11 Fluorescent252-263 761 H 3 Fluorescent 264-276 765 A 8 Fluorescent 287-298 652 D 2Fluorescent 374-379, 392-397 806 A 6 Fluorescent 312-321

Example 7 Recombinant Protein Isolation

After isolation of the desired single plasma cells from Example 4, mRNAwas extracted and reverse transcriptase PCR was conducted to generatecDNA encoding the variable heavy and light chains of the antibodysecreted by each cell. The human variable heavy chain cDNA was digestedwith restriction enzymes that were added during the PCR and the productsof this reaction were cloned into an IgG2 expression vector withcompatible overhangs for cloning. This vector was generated by cloningthe constant domain of human IgG2 into the multiple cloning site ofpcDNA3.1+/Hygro (Invitrogen, Burlington, Ontario, Canada). The humanvariable light chain cDNA was digested with restriction enzymes thatwere added during the PCR reaction and the products of this reactionwere cloned into an IgKappa or IgLamda expression vector with compatibleoverhangs for cloning. This vector was generated by cloning the constantdomain of human IgK or IgL into the multiple cloning site ofpcDNA3.1+/Neo (Invitrogen).

The heavy chain and the light chain expression vectors were thenco-transfected using lipofectamine into a 60 mm dish of 70% confluenthuman embryonal kidney (HEK) 293 cells. The transfected cells secreted arecombinant antibody with the identical specificity as the originalplasma cell for 24 to 72 hours. The supernatant (3 mL) was harvestedfrom the HEK 293 cells and the secretion of an intact antibody wasdemonstrated with a sandwich ELISA to specifically detect human IgG.Specificity was confirmed through binding of the recombinant antibody toαVβ6 using ELISA. The rescued clones secreting antibody that could bindto the target antigen are summarized in Table 7.

TABLE 7 Secretion and Binding Data for the Recombinant Antibodies ParentPlate ID Antibody Plate Row Column ID 68 B 10 49 296 D 10 58 612 G 1 188752 D 12 97 762 D 8 277 766 B 5 133 827 E 12 161 659 F 11 254 761 H 3264 765 A 8 298 652 D 2 374 806 A 6 320

Example 8 Purification of Recombinant Antibodies

For larger scale production of the anti-αVβ6 antibodies, heavy and lightchain expression vectors (2.5 g of each chain/dish) were lipofected intoten 100 mm dishes that were 70% confluent with HEK 293 cells. Thetransfected cells were incubated at 37° C. for 4 days, the supernatant(6 mL) was harvested and replaced with 6 mL of fresh media. At day 7,the supernatant was removed and pooled with the initial harvest (120 mLtotal from 10 plates). The antibodies were purified from the supernatantusing Protein-A Sepharose (Amersham Biosciences, Piscataway, N.J.)affinity chromatography (1 mL). The antibodies were eluted from theProtein-A column with 500 μL of 0.1 M Glycine pH 2.5. The eluate wasdialyzed in PBS pH 7.4 and filter sterilized. The antibodies wereanalyzed by non-reducing SDS-PAGE to assess purity and yield. Proteinconcentration was determined by determining the optical density at 280nm.

Example 9 Structural Analysis of αVβ6 Antibodies

The variable heavy chains and the variable light chains of theantibodies were sequenced to determine their DNA sequences. The completesequence information for the anti-αVβ6 antibodies is provided in thesequence listing with nucleotide and amino acid sequences for each gammaand kappa/lambda chain combination. The variable heavy sequences wereanalyzed to determine the VH family, the D-region sequence and theJ-region sequence. The sequences were then translated to determine theprimary amino acid sequence and compared to the germline VH, D andJ-region sequences to assess somatic hypermutations.

Table 8 is a table comparing the antibody heavy chain regions to theircognate germ line heavy chain region. Table 9 is a table comparing theantibody kappa or lambda light chain regions to their cognate germ linelight chain region.

The variable (V) regions of immunoglobulin chains are encoded bymultiple germ line DNA segments, which are joined into functionalvariable regions (V_(II)DJ_(II) or V_(K)J_(K)) during B-cell ontogeny.The molecular and genetic diversity of the antibody response to αVβ6 wasstudied in detail. These assays revealed several points specific toanti-αVβ6 antibodies.

According the sequencing data, the primary structure of the heavy chainsof sc 298 and sc 374 are similar, but not identical. sc 254 isstructurally different from the other two. It should also be appreciatedthat where a particular antibody differs from its respective germlinesequence at the amino acid level, the antibody sequence can be mutatedback to the germline sequence. Such corrective mutations can occur atone, two, three or more positions, or a combination of any of themutated positions, using standard molecular biological techniques. Byway of non-limiting example, Table 9 shows that the light chain sequenceof sc 298 (SEQ ID NO.: 40) differs from the corresponding germlinesequence (SEQ ID NO.:68) by a Val to Ala mutation (mutation 1) in theFR1 region, via a Leu to Ala mutation (mutation 2) in the CDR1 regionand an Asn to Ser in the FR3 region. Thus, the amino acid or nucleotidesequence encoding the light chain of sc 298 can be modified to changemutation 1 to yield the germline sequence at the site of mutation 1.Further, the amino acid or nucleotide sequence encoding the light chainof mAb sc 298 can be modified to change mutation 2 to yield the germlinesequence at the site of mutation 2. Still further, the amino acid ornucleotide sequence encoding the light chain of mAb sc 298 can bemodified to change mutation 3 to yield the germline sequence at the siteof mutation 3. Still further again, the amino acid or nucleotidesequence encoding the light chain of sc 298 can be modified to changemutation 1, mutation 2 and mutation 3 to yield the germline sequence atthe sites of mutations 1, 2 and 3. Still further again, the amino acidor nucleotide sequence encoding the light chain of sc 298 can bemodified to change any combination of mutation 1, mutation 2 andmutation 3. In another example, heavy chain of sc 264 (SEQ ID NO: 30)differs from it's germline (SEQ ID NO: 55) at position 61. Thus theamino acid or nucleotide sequence encoding the heavy chain of sc 264 canbe modified from a N to Y to yield the germline sequence. Tables 10-13below illustrate the position of such variations from the germline forsc 133, sc 188 and sc 264. Each row represents a unique combination ofgermline and non-germline residues at the position indicated by boldtype. Particular examples of an antibody sequence that can be mutatedback to the germline sequence include: sc 133 where the L at amino acid70 of the heavy chain is mutated back to the germline amino acid of M(referred to herein as sc 133 TMT); sc 133 where the N at amino acid 93of the light chain is mutated back to the germline amino acid of D(referred to herein as sc 133 WDS); and sc 264 where the A at amino acid84 of the light chain is mutated back to the germline amino acid of D(referred to herein as sc 264 ADY).

In one embodiment, the invention features modifying one or more of theamino acids in the CDR regions, i.e., CDR1, CDR2 and/or CDR3. In oneexample, the CDR3 of the heavy chain of an antibody described herein ismodified. Typically, the amino acid is substituted with an amino acidhaving a similar side chain (a conservative amino acid substitution) orcan be substituted with any appropriate amino acid such as an alanine ora leucine. In one embodiment, the sc 264 CDR3, VATGRGDYHFYAMDV (aminoacid residues 100-114 of SEQ ID NO: 30), can be modified at one or moreamino acids. Applicants have already demonstrated that the CDR3 regioncan be modified without adversely affecting activity, i.e., see sc 264RAD where the second G in the CDR3 region is substituted for an A. Othermodifications within the CDR3 region are also envisaged. In anotherembodiment, the sc 133 CDR3 region, RLDV, can be modified at one or moreamino acids including substituting the L for an A and/or the V for an A.Means of substituting amino acids are well known in the art and includesite-directed mutagenesis.

In another embodiment, the invention includes replacing any structuralliabilities in the sequence that might affect the heterogeneity orspecificity of binding of the antibodies of the invention. In oneexample, the antibody sc 264 has an RGD sequence in the CDR3 region thatmight cause cross-reactive binding. Therefore the glycine residue in theRGD can be replaced with an alanine (sc 264 RAD).

TABLE 8  Heavy chain analysis SEQ Chain ID Name NO: V D J FR1 CDR1 FR2CDR2 FR3 CDR3 FR4 49 Germline QVQLVQSGAEVKK GYTFTG WVRQAPG WINPNSGGTRVTMTRDTSISTAYME RL-- WGQGTT PGASVKVSCKAS YYMH QGLEWMG NYAQKFQGLSRLRSDDTAVYYCAR VTVSS sc 133 14 VH1-2 5-12 JH6B QVQLVQSGAEVKK GYTFTGWVRQAPG WINPKSGDT RVTLTRDTSTSTAYME RLDV WGQGTT PGASVKVSCKAS YYMH QGLEWMGNYAQKFQG LSRLRSDDTAVYYCAR VTVSS 50 Germline EVQLVESGGGLVK GFTFSS WVRQAPGSISSSSSYI RFTISRDNAKNSLYLQ --VQLE WGQGTT PGGSLRLSCAAS YSMN KGLEWVSYYADSVKG MNSLRAEDTAVYYCAR RYYYY VTVSS YGMDV sc 320 42 VH3-21 D1-1 JH6BEVQLVESGGGLVK GYTFTN WVRQAPG SISISSSYI RFTISRDNAKNSLYLQ DPVPLE WGQGTTPGGSLRLSCAAS YIMH KGLEWVS YYADSVKG MNSLRAEDTAVYYCAR RRDYY VTVSS YGMDV 51Germline EVQLLESGGGLVQ GFTFSS WVRQAPG AISGSGGST RFTISRDNSKNTLYLQ -VDTAWGQGTT PGGSLRLSCAAS YAMS KGLEWVS YYADSVKG MNSLRAEDTAVYYCAK MVYYG VTVSSMDV sc 58  6 VH3-23 D5-5 JH6B EVQLLESGGGLVQ GFTFSS WVRQAPG AISGSGGSTRFTISRDNSKNTLYLQ GVDTAMV WGQGTT PGGSLRLSCAAS YVMS KGLEWVS YYADSVKGMNSLRAEDTAVYYCAK TYGMDV VTVSS 52 Germline QVQLVESGGGVVQ GFTFSS WVRQAPGVIWYDGSNK RFTISRDNSKNTLYLQ -IAA WGQGTT PGRSLRLSCAAS YGMH KGLEWVAYYADSVKG MNSLRAEDTAVYYCAR R--YYY VTVSS YGMDV sc 298 38 VH3-33 D6-6 JH6BQVQLVESGGGVVQ GFTFSS WVRQAPG VIWYGGSNK RFTISRDNSKNTLYLQ DLAAR WGQGTTPGRSLRLSCAAS YGMH KGLEWVA YYADSVKG MNSLRAEDTAVYYCAR RGDYYY VTVSS YGMDVsc 374 46 VH3-33 D6-6 JH6B QVQLVESGGGVVQ GFTFSS WVRQAPG VIWYDGSNKRFTISRDNSKNTLYLQ TEGIA WGQGTT PGRSLRLSCAAS YGMH KGLEWVA YYADSVKGMNSLRAEDTAVYYCAR ARLYYY VTVSS YGMDV 53 Germline QVQLQESGPGLVK GGSISSWIRQHPG YIYYSGSTY RVTISVDTSKNQFSLK --GIAA WGQGTT PSQTLSLTCTVS GGYYWSKGLEWIG YNPSLKS LSSVTAADTAVYYCAR AG--YY VTVSS YYYGMDV sc 254 26 VH4-31D6-13 JH6B QVQLQESGPGLVK GGSISS WIRQHPG YIYYSGSTY RVTISVDTSKNQFSLKYRGPAA WGQGTT PSQTLSLTCTVS GGYYWS KGLEWIG YNPSLKS LSSVTAADTAMYYCARGRGDFY VTVSS YFGMDV 54 Germline QVQLQESGPGLVK GGSISS WIRQHPG YIYYSGSTYRVTISVDTSKNQFSLK ---ITI WGQGTL PSQTLSLTCTVS GGYYWS KGLEWIG YNPSLKSLSSVTAADTAVYYCAR FGVFDY VTVSS sc 49  2 VH4-31 D3-3 JH4B QVQLQESGPGLVKGGSIRS WIRQHPG NIYYSGSTY RITISVATSRNQFSLK GGAITIF WGQGTL PSQTLSLTCTVAGDYYWS KGLEWIG YNPSLKS LTSVTAADTAVYYCAR GVFDY VTVSS 55 GermlineQVQLQESGPGLVK GGSISS WIRQHPG YIYYSGSTY RVTISVDTSKNQFSLK VAT--- WGQGTTPSQTLSLTCTVS GGYYWS KGLEWIG YNPSLKS LSSVTAADTAVYYCAR YYYYYG VTVSS MDVSc 264 30 VH4-31 D4-17 JH6B QVQLQESGPGLVK GGSISS WIRQHPG YIYYSGRTYRVTISVDTSKNQFSLK VATGRG WGQGTT PSQTLSLTCTVS GGYYWS KGLEWIG NNPSLKSLSSVTAADTAVYYCAR DYHFYA VTVSS MDV 56 Germline QVQLQESGPGLVK GGSISSWIRQHPG YIYYSGSTY RVTISVDTSKNQFSLK ---LRY WGQGTT PSQTLSLTCTVS GGYYWSKGLEWIG YNPSLKS LSSVTAADTAVYYCAR YYYYGM VTVSS DV Sc 188 22 VH4-31 D4-23JH6B QVQLQESGPGLVK GGSISS WIRQHPG YIYYSGSTS RVTISVDTSKKQFSLN EGPLRGWGQGTT PSQTLSLTCTVS GVYYWT NGLEWIG YNPSLKS LTSVTAADTAVYYCAR DYYYGL VTVSSDV 57 Germline EVQLVQSGAEVKK GYSFTS WVRQMPG IIYPGDSDT QVTISADKSISTAYLQ---SSG WGQGTM PGESLKISCKGS YWIG KGLEWMG RYSPSFQG WSSLKASDTAMYYCARYYYAFDI VTVSSA Sc 97  10 VH5-51 D3-22 JH3B EVQLVQSGAEVKK GYSFTS WVRQMPGIIYPGDSDT QVILSADKSISTAYLQ HDESSGY WGQGTM PGESLKISCKGS YWIG KGLEWMGRYSPSFQG WSSLKASDTAMYYCAR YYVFDI VTVSSA 58 Germline EVQLVQSGAEVKK GYSFTSWVRQMPG IIYPGDSDT QVTISADKSISTAYLQ -----G WGQGTT PGESLKISCKGS YWIGKGLEWMG RYSPSFQG WSSLKASDTAMYYCAR MDV VTVSS Sc 277 34 VH5-51 D3-10 JH6BEVQLVQSGAEVKK GYSFPS WVRQMPG IIYPGDSDT QVTISADKSISTAYLQ HPMEDG WGQGTTPGESLKISCKGS YWIG KGLEWMG RYSPSFQG WSSLKASDTAMYYCAR MDV VTVSS 59Germline EVQLVQSGAEVKK GYSFTS WVRQMPG IIYPGDSDT QVTISADKSISTAYLQ-GIAAAG- WGKGTT PGESLKISCKGS YWIG KGLEWMG RYSPSFQG WSSLKASDTAMYYCARYYYGMDV VTVSSA Sc 161 18 VH5-51 D6-13 JH6C EVQLVQSGAEVKK GYSFTS WVRQMPGIIYPGDSDT QVTISADKSISTAYLQ HGIAAAGF WGQGTT PGESLKISCKGS YWIG KGLEWMGRYSPSFQG WSSLKASDTAMYYCAR YYYYMDV VTVSSA

TABLE 9  Light chain analysis SEQ Chain ID V Name NO: Kappa J FR1 CDR1FR2 CDR2 FR3 CDR3 J 60 Germline DIVMTQTPLSLS KSSQSLLH WYLQKPGQ EVSNGVPDRFSGSGSGTDFT MQSIQL FGQGTK VTPGQPASISC SDGKTYLY PPQLLIY RFSLKISRVEAEDVGVYYC PWT VEIK Sc 254 28 A2 JK1 DIVMTQTPLSLS KSSQSLLNWYLQKPGQ EVSN GVPDRFSGSGSGTDFT MQGIQL FGQGTK VTPGQPASIFC SDGKTYLCPPQLLIY RFS LKISRVEAEDVGVYYC PWAF VEIK 61 Germline EIVLIQSPGILS RASQSVSSWYQQKPGQ GASS GIPDRFSGSGSGTDFT QQYGSS FGQGTK LSPGERATLSC SYLA APRLLIYRAT LTISRLEPEDFAVYYC PWT VEIK sc 188 24 A27 JK1 EIVLTQSPGTLS RAGQTISSWYQQKPGQ GASS GIPDRFSGSGSGTDFT QQYGSS FGQGTK LSPGERATLSC RYLA APRPLIYRAT LTISRLEPEDFAVYYC PRT VEIK sc 374 48 A27 JK1 EIVLTQSPGTLS RASQSVSSWYQQKPGQ GASS DIPDRFSGSGSGTDFT QQYGSS FGQGTK LSPGERATLSC SYLA APRLLIYRAT LTISRLEPEDFAVYYC PWT VEIK 62 Germline EIVLTQSPGILS RASQSVSS WYQQKPGQGASS GIPDRFSGSGSGTDFT QQYGSS FGQGTK LSPGERATLSC SYLA APRLLIY RATLTISRLEPEDFAVYYC PYT LEIK Sc 49  4 A27 JK2 EIVLTQSPGTLS RASQSVSSWYQQKPGQ GASS GIPDRFSGSGSGTDFT QQYGSS FGQGTK LSPGERATLSC SYLA APRLLIYRAT LTISRLEPEDFAVYYC PCS LEIK 63 Germline EIVLTQSPGILS RASQSVSS WYQQKPGQGASS GIPDRFSGSGSGTDFT QQYGSS FGPGTK LSPGERATLSC SYLA APRLLIY RATLTISRLEPEDFAVYYC PET VDIKR Sc 161 20 A27 JK3 EIVLTQSPGTLS RASQNVNRWYQQKPGQ GTSN GIPDRFSGSGSGTDFT QQCGSL FGPGTK LSPGERASLSC NYLV APRLLIYRAT LTISRLEPEDFAVYYC PFT VDIKR 64 Germline QSVLTQPPSVSA SGSSSNIGWYQQLPGT DNNK GIPDRFSGSKSGTSAT GTWDSS FGTGTK APGQKVTISC NNYVS APKLLIYRPS LGITGLQTGDEADYYC LSA-YV VTV sc 133 16 V1-19 JL1 QSVLTQPPSVSASGSSSNIG WYQQLPGT DNNK GIPDRFSGSKSGTSAT GTWNSS FGTGTK APGQKVTISC NNYVSAPKLLIY RPS LGITGLQTGDEADYYC LSAGYV VTV 65 Germline QSVLTQPPSVSASGSSSNIG WYQQLPGT DNNK GIPDRFSGSKSGTSAT GTWDSS FGGGTK APGQKVTISC NNYVSAPKLLIY RPS LGITGLQTGDEADYYC LSAVV LTVL sc 320 44 V1-19 JL2 QSVLTQPPSMSASGSSSNIG WYQQLPGT DNNK GIPDRFSGSKSGTSAT GTWDSS FGGGTK APGQKVTISC NNYVSAPKLLIY RPS LGITGLQTGDEADYYC LSAGV LTVL 66 Germline SYELTQPPSVSVSGDALPKK WYQQKSGQ EDSK GIPERFSGSSSGTMAT YSTDSS FGGGTK SPGQTARITC YAYAPVLVIY RPS LTISGAQVEDEADYYC GNHVV LTVL sc 277 36 V2-7 JL2 SYELTQPPSVSVSGDALPKK WYQQKSGQ DDNK GIPERFSGSSSGTMAT YSTDSS FGGGTK SPGQTARITC YAFAPVLVIY RPS LTISGAQVEDEADYYC GRP LTVL sc 97  12 V2-7 JL2 SYELTQPPSVSVSGDALPKK WYQQKSGQ EDIK GIPERFSGSSSGTMAT YSTDSS FGGGTK SPGQTARITC YAYAPVLVIY RPS LTISGAQVEDEADYYC GNEWVF LTVL 67 Germline SYELTQPPSVSVSGDALPKK WYQQKSGQ EDSK GIPERFSGSSSGTMAT YSTDSS FGGGTK SPGQTARITC YAYAPVLVIY RPS LTISGAQVEDEADYYC GNHVV LTVL sc 58  8 V2-7 JL3 SYELTQPPSVSVSGDALPKK WYQQKSGQ DDSK GIPERFSGSSSGTMAT YSTDSS FGGGTK SPGQTARITC YAYAPVLVIY RPS LTISGAQVEDEADYYC GRIP LTVL 68 Germline SSELTQDPAVSV QGDSLRSYWYQQKPGQ GKNN GIPDRFSGSSSGNTAS NSRDSS FGGGTK ALGQTVRITC YAS APVLVIY RPSLTITGAQAEDEADYYC GNHVV LTVL sc 298 40 V2-13 JL2 SSELTQDPVVSV QGDSLRSYWYQQKPGQ GKNN GIPDRFSGSNSGNTAS NSRDSS FGGGTK ALGQTVRITC YLS APVLVIY RPSLTITGAQAEDEADYYC GNHL LTVL 69 Germline SYELTQPSSVSV SGDVLAKK WFQQKPGQKDSE GIPERFSGSSSGTTVT YSAADN FGGGTK SPGQTARITC YAR APVLVIY RPSLTISGAQVEDEADYYC NVV LTVL sc 264 32 V2-19 JL2 SYELTQPSSVSV SGDVLAKKWFHQKPGQ KDSE GIPERFSGSSSGTTVT YSAADN FGGGTK SPGQTARITC SAR APVLVIY RPSLTISGAQVEDEAAYYC NLV LTVL

TABLE 10 Exemplary Mutations of sc 133 Heavy Chain (SEQ ID NO: 14) toGermline (SEQ ID NO: 49) at the indicated Residue Number 54 57 70 76 N GM T N G L I N G L T N D M I N D L I N D M T N D L T K G M I K G M T K GL I K G L T K D M I K D L I K D M T

TABLE 11 Exemplary Mutations of sc 188 Light Chain (SEQ ID NO: 24) toGermline (SEQ ID NO: 61) at the indicated Residue Number 26 28 29 32 47G S V S L G S V S P G S V R P G S V R L G S V R L G S V S P G S I R P GS I R L G T V R L G T V S P G T V S L G T I R P G T I R L G T I S L S SV S P S S V R P S S V R L S S V R L S S V S P S S I R P S S I R L S T VR L S T V S P S T V S L S T I R P S T I R L S T I S L

TABLE 12 Exemplary Mutations of sc 188 Heavy Chain (SEQ ID NO: 22) toGermline (SEQ ID NO: 56) at the indicated Residue Number 33 37 45 60 7883 85 G S K Y N K S G S K Y N K T G S K Y N N S G S K Y N N T G S K Y KN S G S K Y K N T G S K Y K K S G S K Y K K T G S K S N K S G S K S N KT G S K S N N S G S K S N N T G S K S K N S G S K S K N T G S K S K K SG S K S K K T G S N Y N K S G S N Y N K T G S N Y N N S G S N Y N N T GS N Y K N S G S N Y K N T G S N Y K K S G S N Y K K T G S N S N K S G SN S N K T G S N S N N S G S N S N N T G S N S K N S G S N S K N T G S NS K K S G S N S K K T V S K Y N K S V S K Y N K T V S K Y N N S V S K YN N T V S K Y K N S V S K Y K N T V S K Y K K S V S K Y K K T V S K S NK S V S K S N K T V S K S N N S V S K S N N T V S K S K N S V S K S K NT V S K S K K S V S K S K K T V S N Y N K S V S N Y N K T V S N Y N N SV S N Y N N T V S N Y K N S V S N Y K N T V S N Y K K S V S N Y K K T VS N S N K S V S N S N K T V S N S N N S V S N S N N T V S N S K N S V SN S K N T V S N S K K S V S N S K K T G I K Y N K S G I K Y N K T G I KY N N S G I K Y N N T G I K Y K N S G I K Y K N T G I K Y K K S G I K YK K T G I K S N K S G I K S N K T G I K S N N S G I K S N N T G I K S KN S G I K S K N T G I K S K K S G I K S K K T G I N Y N K S G I N Y N KT G I N Y N N S G I N Y N N T G I N Y K N S G I N Y K N T G I N Y K K SG I N Y K K T G I N S N K S G I N S N K T G I N S N N S G I N S N N T GI N S K N S G I N S K N T G I N S K K S G I N S K K T V I K Y N K S V IK Y N K T V I K Y N N S V I K Y N N T V I K Y K N S V I K Y K N T V I KY K K S V I K Y K K T V I K S N K S V I K S N K T V I K S N N S V I K SN N T V I K S K N S V I K S K N T V I K S K K S V I K S K K T V I N Y NK S V I N Y N K T V I N Y N N S V I N Y N N T V I N Y K N S V I N Y K NT V I N Y K K S V I N Y K K T V I N S N K S V I N S N K T V I N S N N SV I N S N N T V I N S K N S V I N S K N T V I N S K K S V I N S K K T

TABLE 13 Exemplary Mutations of sc 264 Light Chain (SEQ ID NO: 32) toGermline (SEQ ID NO: 69) at the indicated Residue Number 31 36 84 Y H AY H D Y Q A S H D S Q D S Q A

Example 10 Potency Determination of αVβ6 Antibodies

To discriminate antibodies based on their ability to prevent theadhesion of HT29 cells to TGFβLAP, the following adhesion assay wasperformed.

Nunc MaxiSorp (Nunc) plates were coated overnight with 50 μL of 10 μg/mlTGF Beta1 LAP (TGFβLAP), and pre-blocked with 3% BSA/PBS for 1 hourprior to the assay. HT29 cells grown to the optimal density were thenpelleted and washed twice in HBBS (with 1% BSA and without Mn²⁺), afterwhich the cells were then resuspended in HBSS at 30,000 cell per well.The coating liquid was removed from the plates, which were then blockedwith 100 μL 3% BSA at room temperature for 1 hour and thereafter washedtwice with PBS.

Antibody titrations were prepared in 1:3 serial dilutions in a finalvolume of 30 μL and at two times the final concentration. Care was takento ensure that the PBS concentration in the control wells matched thePBS concentration in the most dilute antibody well. 30 μL of cells wereadded to each well, and the cells were incubated in the presence of theantibodies at 4° C. for 40 minutes in a V-bottom plate. Thecell-antibody mixtures were transferred to the coated plate and theplate was incubated at 37° C. for 40 minutes. The cells on the coatedplates were then washed four times in warm HBSS, and the cells werethereafter frozen at −80° C. for 15 minutes. The cells were allowed tothaw at room temperature, and then 100 μL of CyQuant dye/lysis buffer(Molecular Probes) was added to each well according to themanufacturer's instructions. Fluorescence was read at an excitationwavelength of 485 nm and an emission wavelength of 530 nm. An estimatedIC₅₀ value for each mAb was calculated based on the maximal and minimalamount of cell adhesion possible in the assay, as determined by positiveand negative control wells. The results for twelve antibodies aresummarized in Table 14.

TABLE 14 Adhesion Assay Results (Estimated IC₅₀ Values) n = 1 (ng/mL) n= 2(ng/mL) n = 3 (ng/mL) sc 049 >5000 >5000 >5000 sc 058 4065 2028 3259sc 097 1006 281 536 sc 133 25 16 30 sc 161 2408 137 ND sc 188 41 26 NDsc 254 63 37 37 sc 264 26 14 18 sc 277 1455 540 720 sc 298 29 25 33 sc320 648 381 415 sc 374 277 300 549 Positive 226 185 286 Control 2077Z

Example 11 Competition Assay

To establish that the antibodies were specifically capable of blockingαVβ6 integrin binding to soluble TGFβLAP, a competition assay was runwith the purified antibodies to measure their ability to bind to αVβ6and block its binding to a GST-LAP peptide.

Medium binding 96-well plates (Costar, catalog #3368) were coated with50 μL/well of 10 μg/ml GST-LAP in PBS and 0.05% sodium azide, andincubated overnight at 4° C. The plates were then washed three timesusing 300 μL/well of assay diluent (1% milk in TBS (50 mM Tris, 50 mMNaCl, 1 mM MgCl₂ and 1 mM CaCl₂, pH 6.9), after which the plates wereblocked using 300 μL/well 5% milk in TBS and incubated for 30 minutes atroom temperature. The mAbs (in 1:3 serial dilutions ranging from 10μg/ml to 0.01 μg/ml) were incubated overnight with αVβ6 (250 ng/ml inassay diluent containing 0.05% sodium azide). The following day, 50μL/well of the pre-incubated primary antibody was transferred to theGST-LAP peptide-coated plate and incubated for one hour at roomtemperature. The wells were then washed three times using 300 μL/well ofassay diluent. Then, to detect the amount of αVβ6 bound to the plates,mAb 2075 (Chemicon) was added at a concentration of 1 μg/ml in assaydiluent (50 μL/well) and incubated for one hour at room temperature. Thewells were then washed three times using 300 μL/well of assay diluent,and incubated with goat anti-mouse IgG Fc-peroxidase at 400 ng/ml inassay diluent (50 μL/well) for one hour at room temperature. The wellswere then washed three times using 300 μL/well of assay diluent, anddeveloped using 1-step TMB (Neogen) at a total volume of 50 μL/well.After 15 minutes, the developing reaction was quenched with 50 μL/wellof 1N Hydrochloric acid. The plates were read at 450 nm, and the resultsfor five of the antibodies are summarized in FIG. 1, which shows thatthe antibodies were able to inhibit αVβ6 binding to GST-LAP.

Example 12 Cross-Reactivity to αvβ3 or αVβ5 Integrins

To establish that the antibodies were functional only against αVβ6integrin and not αVβ3 or αVβ5 integrins, the following assay wasperformed to test the ability of the antibodies to inhibit the adhesionof A375M cells to an osteopontin peptide.

Assay plates were coated with osteopontin peptide. Two fragments ofosteopontin were used: OPN 17-168 and OPN 17-314. Assay plates werepre-blocked with 3% BSA/PBS for one hour prior to the assay. The A375Mcells were removed from a culture flask, pelleted and washed twice withHBSS containing 1% BSA and 1 mM Ca²⁺ and 1 mM Mg²⁺. The cells were thenresuspended in HBSS at a concentration of 30,000 cells per well. Thecoating liquid containing the osteopontin fragments was removed, and theplates were blocked with 100 μL of 3% BSA for one hour at roomtemperature. The coated plates were washed twice with HBSS containing 1%BSA. Antibody titrations were prepared in 1:4 serial dilutions in afinal volume of 30 μL and at twice the final concentration. Theresuspended cells were added to the wells containing the titratedantibody in a V-bottom plate, and the cells and antibodies wereco-incubated at 4° C. for 40 minutes. The cell-antibody mixture was thentransferred to the coated plate, which was thereafter incubated at 37°C. for 40 minutes. The cells on the coated plates were next washed fourtimes in warm HBSS, and the cells in the plates were then frozen at −80°C. for 15 minutes. The cells were allowed to thaw at room temperature,and then 100 μL of CyQuant dye/lysis buffer (Molecular Probes) was addedto each well according to the manufacturer's instructions. Fluorescencewas read at an excitation wavelength of 485 nm and an emissionwavelength of 530 nm.

The results for five of the antibodies are summarized in Table 15. Acommercially available αV integrin specific antibody was used as apositive control in this assay and exhibited about 90% inhibition ofadhesion. A commercially available αVβ6 antibody served as a negativecontrol in this assay for adhesion to αVβ3 or αVβ5 integrins. Allantibodies were tested at a concentration of 5 μg/ml and none of thetest antibodies could block adhesion to αVβ3 or αVβ5 integrins.

TABLE 15 Cross-Reactivity to αVβ3 or αVβ5 Integrins Percent Antibody IDInhibition sc 133 3 sc 188 −2 sc 254 −5 sc 264 3 sc 298 9 αV Control 89αVβ6 Control 11 Human IgG Control 3 Mouse IgG Control 5

Example 13 Cross-Reactivity to α4β1 Integrin

To establish that the antibodies were functional only against the αVβ6integrin and not the α4β1 integrin, an assay was performed to test theability of the antibodies to inhibit the adhesion of J6.77 cells to theCS-1 peptide of fibronectin. The assay was performed as described inExample 12 above, with the exception that J6.77 cells were used forbinding and the CS-1 peptide of fibronectin was used to coat the assayplates.

The results for 11 of the antibodies are summarized in Table 16. Acommercially available β1 integrin specific antibody was used as apositive control in this assay and exhibited 97% inhibition of adhesion.A commercially available αVβ6 specific antibody served as a negativecontrol in this assay for adhesion to α4β1. All antibodies were used at5 μg/ml and none of the test antibodies could block adhesion to α4β1.

TABLE 16 Cross-Reactivity to α4β1 Integrin Percent Antibody at 5 ug/mlInhibition sc 58 −14 sc 97 −7 sc 133 −15 sc 161 12 sc 188 −10 sc 254 0sc 264 −8 sc 277 −17 sc 298 −7 sc 320 −8 sc 374 −8 Human IgG1 −6 HumanIgG2 −9 Anti-beta1 integrin antibody 97 Anti-αVβ6 integrin antibody −15No CS-1 or antibody on plates 12 CS-1 fragment coated 10 plates withoutantibody

Example 14 Cross-Reactivity to α5β1 Integrin

To establish that the antibodies were functional only against the αVβ6integrin and not the α5β1 integrin, an adhesion assay was performed totest the ability of the antibodies to inhibit the adhesion of K562 cellsto fibronectin.

Assay plates were coated with the FN9-10 peptide of fibronectin at aconcentration of 12.5.1 g/mL. Assay plates were pre-blocked with 3%BSA/PBS for one hour prior to the assay. The K562 cells were removedfrom a culture flask, pelleted and washed twice with HBSS containing 1%BSA and 1 mM Mn²⁺. The cells were then resuspended in HBSS at aconcentration of 30,000 cells per well. The coating liquid containingthe osteopontin fragments was removed, and the plates were blocked with100 μL of 3% BSA for one hour at room temperature. The coated plateswere washed twice with HBSS containing 1% BSA. Antibody titrations wereprepared in 1:4 serial dilutions in a final volume of 30 μL and at twicethe final concentration. The resuspended cells were added to the wellscontaining the titrated antibody in a V-bottom plate, and the cells andantibodies were co-incubated at 4° C. for 60 minutes. The cell-antibodymixture was then transferred to the coated plate, which was thereafterincubated at 37° C. for 40 minutes. The cells on the coated plates werenext washed four times in warm HBSS, and the cells in the plates werethen frozen at −80° C. for 15 minutes. The cells were allowed to thaw atroom temperature, and then 100 μL of CyQuant dye/lysis buffer (MolecularProbes) was added to each well according to the manufacturer'sinstructions. Fluorescence was read at an excitation wavelength of 485nm and an emission wavelength of 530 nm.

The results for five of the antibodies are summarized in Table 17. Testantibodies were compared to a commercially available α5β1 antibody as apositive control and an αVβ6 specific antibody as a negative control.None of the test antibodies were able to block adhesion in the assay atthe 5 μg/ml concentration used in this assay.

TABLE 17 Cross-Reactivity to α5β1 Integrin Percent Antibody IDInhibition sc 133 −12 sc 188 5 sc 254 −9 sc 264 −4 sc 298 2 αVβ6 Control7 α5β1 Control 78 Human IgG 11 Control

Example 15 Cross-Reactivity to Murine and Cynomolgus αVβ6 Integrin

In order to determine whether the antibodies exhibited cross-reactivityto mouse αVβ6 or Cynomolgus αVβ6, the following assay was performed.

Cross-reactivity of the mAbs to macaque and mouse αVβ6 was tested on thepurified mAbs using FACS analysis on HEK-293 cells transientlytransfected with cynomolgus or mouse αV, β6, or αVβ6. Approximately 48hours after transfection, the cells were collected and resuspended inFACS buffer to reach a final concentration of approximately 50,000 cellsin 100 μL.

Approximately 100,000 cells total, were used in each reaction asfollows. 200 μL of 293 cells were dispensed into a V-bottom plate. Thecells in the plate were pelleted at 1500 rpm for 3 minutes, and thenresuspended in 100 μL FACS buffer. The pelleting step was repeated, andthe FACS buffer supernatant was removed. The purified mAbs, or controlprimary antibodies were added in a volume of 50 μL and the cells wereresuspended. Primary antibody controls were murine αVβ6 (Cat#MAB2077z,Chemicon) and anti-αV and anti-β6 recombinants. The plate was incubatedon ice for 45 minutes, after which 100 μL FACS buffer was added todilute the primary antibody. The cells were then pelleted bycentrifuging at 1500 rpm for 3 minutes, and the pellet was resuspendedin 100 μL FACS buffer. The pelleting step was repeated, and the FACSbuffer supernatant was removed. Cells were then resuspended in theappropriate secondary antibody (5 μg/ml) with 7AAD dye (10 μg/ml), andstained on ice for 7 minutes. Then 150 μL of FACS buffer was added andthe cells were pelleted at 1500 rpm for 3 minutes, after which the cellswere washed in 100 μL FACS buffer, pelleted, and then resuspended in 250μL buffer and added to FACS tubes. Samples were analyzed on a highthroughput FACS machine and analyzed using Cell Quest Pro software.

The results are summarized in Table 18, and demonstrate that mAb sc 133and mAb sc 188 were clearly cross-reactive with mouse and CynomolgusαVβ6 and β6. mAb sc 254 appeared to cross-react with β6, αV, and αVβ6.mAbs sc 264 and 298 had high levels of binding to parental cells makingspecies cross-reactivity difficult to discern.

TABLE 18 Cross-Reactivity with Mouse and Cynomolgus αVβ6 Mouse MouseMouse Cynomolgus Cynomolgus Cynomolgus Antibodies Parental alphaV beta6alphaVbeta6 alphaV beta6 alphaVbeta6 Cells alone 0 0 0 0 1 0 0 Gt anti 00 0 0 0 0 0 Mouse anti 0 1 11 45 0 5 20 alphaVbeta6 anti alphaV 68 68 6359 68 69 67 anti beta6 0 0 0 0 0 0 0 Gt anti 0 0 0 0 0 0 0 Human Human 01 0 1 1 1 0 IgG1 sc.133 2 4 19 49 5 10 28 sc.188 1 3 29 51 2 17 27sc.254 8 13 21 50 16 19 26 sc.264 74 71 68 63 70 75 54 sc.298 49 45 5253 48 52 38 Data represent percent of cells shifted

Example 16 Internalization Assay

The internalization of the antibodies was tested using a K562 cell linethat stably expressed human αVβ6. Internalization of the purifiedantibodies was compared to a commercially available αVβ6 antibody thatwas not internalized in this assay.

The results are summarized in Table 19.

TABLE 19 Summary of the Internalization Assay Concentration PercentAntibody (ug/mL) Internalization sc133 10 28% sc133 1 30% sc 188 10 38%sc 188 1 34% sc 254 10 49% sc 254 1 39% sc 264 10 76% sc 264 1 77% sc298 10 28% sc 298 1 26%

Example 17 High Resolution Biacore Analysis

High resolution Biacore analysis using a soluble αVβ6 protein to bindantibodies immobilized on CM5 chips was performed for 5 of the αVβ6antibodies to estimate their affinity for soluble antigen.

The Biacore analysis was performed as follows. A high-density goat αhuman IgG antibody surface over two CM5 Biacore chips was prepared usingroutine amine coupling. Each mAb was diluted in degassed HBS-P runningbuffer containing 100 μg/ml BSA, 1 mM MgCl₂, and 1 mM CaCl₂ to aconcentration of approximately 1 μg/mL. More precisely, mAb sc 133 wasdiluted to 0.98 μg/mL, mAb sc 188 was diluted to 0.96 g/mL, mAb sc 264was diluted to 0.94 μg/mL, mAb sc 254.2 was diluted to 0.87 μg/mL, andmAb sc 298 was diluted to 1.6 μg/mL. Then, a capture level protocol wasdeveloped for each mAb by capturing each mAb over a separate flow cellat a 10 μL/min flow rate at the concentrations listed above. mAbs sc133, sc 298, and sc 254.2 were captured for 30 seconds while mAbs sc 188and sc 264 were captured for 1 minute. A 4-minute wash step at 50 μL/minfollowed to stabilize the mAb baseline.

Soluble αVβ6 was injected for 4 minutes at a concentration range of116-3.6 nM for mAbs sc 133, sc 188, sc 264, and sc 298, and 233-3.6 nMfor mAb sc 254.2, with a 2× serial dilution for each concentrationrange. A 10-minute dissociation followed each antigen injection. Theantigen samples were prepared in the HBS-P running described above. Allsamples were randomly injected in triplicate with several mAbcapture/buffer inject cycles interspersed for double referencing. Thehigh-density goat α mouse antibody surfaces were regenerated with one18-second pulse of 146 mM phosphoric acid (pH 1.5) after each cycle at aflow rate of 100 μL/min. A flow rate of 50 μL/min was used for allantigen injection cycles.

The data were then fit to a 1:1 interaction model with the inclusion ofa term for mass transport using CLAMP. The resulting binding constantsare listed in Table 20. The mAbs are listed from highest to lowestaffinity.

TABLE 20 Affinity Determination Results for Cloned and Purified mAbsDerived from High Resolution Biacore ™. Antibody R_(max) k_(a) (M⁻¹s⁻¹)k_(d) (s⁻¹) K_(D) (nM) sc 264 96 5.85 × 10⁴ 3.63 × 10⁻⁴ 6.2 sc 298 775.65 × 10⁴ 1.18 × 10⁻³ 21.0 sc 188 76 4.52 × 10⁴ 9.56 × 10⁻⁴ 21.2 sc 13396 5.73 × 10⁴ 1.89 × 10⁻³ 33.0 sc 254.2 53, 45 5.73 × 10⁴ 5.64 × 10⁻⁴34.9

Example 18 Binding Affinity Analysis Using FACS

As an alternative to Biacore, FACS analysis was also used to estimatethe binding affinity of one of the antibodies to K562 cells that stablyexpress the human αVβ6 antigen. The amount of antigen was titrated togenerate a binding curve and estimate the binding affinity to theantigen.

K562 cells expressing αVβ6 were resuspended in filtered HBS buffercontaining 1 mM of MgCl₂ and 1 mM of CaCl₂ at a concentration ofapproximately 6 million cells/mL. The cells were kept on ice. PurifiedmAb sc 188 was serially diluted by a factor of 1:2 in HBS across 11wells in a 96-well plate. The 12^(th) well in each row contained bufferonly. Titrations were done in triplicate. Additional HBS and cells wereadded to each well so that the final volume was 300 μL/well and eachwell contained approximately 120,000 cells. The final molecularconcentration range for mAb sc 188 was 4.9-0.019 nM. The plates wereplaced into a plate shaker for 5 hours at 4° C., after which the plateswere spun and washed three times with HBS, following which, 200 μL of131 nM Cy5 goat α-human polyclonal antibody (Jackson Laboratories,#109-175-008) were added to each well. The plates were then shaken for40 minutes at 4° C., and thereafter were spun and washed once againthree times with HBS. The Geometric Mean Fluorescence (GMF) of 20,000“events” for each mAb concentration was recorded using a FACSCaliburinstrument, and the corresponding triplicate titration points wereaveraged to give one GMF point for each mAb concentration. A plot of theaveraged GMF as a function of molecular mAb concentration with Scientistsoftware was fit nonlinearly using the equation:

$F = {{P^{\prime}\frac{\left( {K_{D} + L_{T} + {n \cdot M}} \right) - \sqrt{\left( {K_{D} + L_{T} + {n \cdot M}} \right)^{2} - {4{n \cdot M \cdot L_{T}}}}}{2}} + B}$

In the above equation, F=geometric mean fluorescence, L_(T)=totalmolecular mAb concentration, P′=proportionality constant that relatesarbitrary fluorescence units to bound mAb, M=cellular concentration inmolarity, n=number of receptors per cell, B=background signal, andK_(D)=equilibrium dissociation constant. For mAb sc 188 an estimate forK_(D) is obtained as P′, n, B, and K_(D) are allowed to float freely inthe nonlinear analysis.

The resulting plot with its nonlinear fits (red line) is shown in FIG.2. Table 21 lists the resulting K_(D) for mAb sc 188 along with the 95%confidence interval of the fit. These results for mAb sc 188 indicatebinding to one type of receptor.

Binding affinity for sc 188 as determined by FACS was significantlytighter than as determined by Biacore (Example 17). There are at least 2possible explanations for the difference in K_(D) values for sc 188. Thefirst reason is that the two assays used different forms of the antigenfor the measurement—Biacore used soluble antigen and the FACs analysisused a cell-bound form of the antigen. The second reason is that theantibodies that were tested were raised against the cell-bound form ofthe antigen and may not bind with as high an affinity to the solubleantigen as they do to the cell-bound antigen.

TABLE 21 Binding Affinity Analysis Using FACS Antibody K_(D) (pM) 95% CI(pM) sc 188 51.9 ±22.7

Example 19 CDC Assay

The purified antibodies described in the examples above are of the IgG1isotype and can have effector function. In order to determine theability of these antibodies to mediate complement-dependent cytotoxicity(CDC), the following assay was performed using 293 cells stablyexpressing αVβ6 (293-10A11) and parental 293 cells (293F).

For calcein staining of cells, aliquots of approximately 25×10e6 each ofHT29, 293-10A11, and 293F cells were individually resuspended in 3 mlserum-free RPMI media. 45 μL of 1 mM calcein was then added to each 3 mlsample of cells, and the samples were incubated at 37° C. for 45minutes. The cells were centrifuged at 1200×RPM for 3 minutes, thesupernatant was discarded and the cells were resuspended in eachrespective cell line's culture media. The centrifugation step wasrepeated and the cells were resuspended to give a final concentration ofabout 100,000 cells in 50 μL media.

Serial 1:2 dilutions of each antibody were prepared in a v-bottom96-well plate, with concentrations ranging from 20 μg/ml to 0.625 μg/mlin a volume of 50 μL. Then, 100,000 of the cells prepared as describedabove were added in a volume of 50 μL to the antibody-containing plates,and the resulting mixture was incubated on ice for two hours. Followingthe incubation, the cells were pelleted, and the supernatant wasdiscarded. The cells were resuspended in 100 μL of their respectivemedia containing 10% human sera (ABI donor #27), and incubated at 37° C.for 30 minutes. The cells were then centrifuged, and 80 μL of thesupernatant was transferred to a FMAT plate. The plate was read on aTecan reader using an excitation wavelength of 485 nm and an emissionwavelength of 530 nm.

The results are summarized in FIGS. 3A-3E, and demonstrate that eachpurified antibody tested is capable of mediating CDC in 293 cells stablyexpressing αVβ6 integrin.

Example 20 Site-Directed Mutagenesis

One of the antibodies (sc 264) prepared from the immunizations(Example 1) showed strong functional blocking activity in vitro in theTGFβLAP binding inhibition assay (see Example 4), but exhibitedcross-reactive binding to non-αVβ6 expressing cell lines (see Example15). This antibody, sc 264, has an RGD sequence in the CDR3 region,which is presumed to be responsible for the cross-reactive binding.Therefore, site-directed mutagenesis was used to replace the glycineresidue in the RGD with an alanine (sc 264 RAD).

A second antibody (sc 188) has a glycosylation site within the FR3region. This site was eliminated through site-directed mutagenesis witha substitution from NLT to KLT (sc 188 SDM). The mutated versions ofthese two antibodies were then expressed and purified as described inExamples 7 and 8, and the purified antibodies were analyzed as describedin the following examples.

Example 21 Binding Assay to Test Cross-Reactive Binding of MutantAntibodies

A binding assay was performed to test whether the cross-reactive bindingobserved in Example 15 was reduced because of site-directed mutagenesisof sc 264. Binding of the antibodies was analyzed on 293T and 293F celllines to test whether removing the RGD site from sc 264 would result indecreased binding compared with the original antibody.

293T and 293F cells were spun down after collection and resuspended inHBSS with 1% BSA and 1 mM CaCl₂ and 1 mM MgCl₂ (wash buffer), so that atleast 150,000 cells were used in each reaction. Cells were dividedbetween reactions in a V-bottom 96-well plate (Sarstedt), and the cellsin the plate were pelleted at 1500 rpm for 3 minutes, after which theHBSS supernatant was removed. The primary antibody was added at theconcentration indicated in Table 19 in a volume of 50 μL, and the cellswere resuspended and thereafter incubated on ice for 60 minutes. Afterincubation, the cells were pelleted by centrifugation at 1500 rpm for 3minutes, resuspended in 100 μL wash buffer, and then pelleted again.Cells were then resuspended in the appropriate secondary antibody at 2μg/ml with 10 μg/ml 7AAD, and stained on ice for 7 minutes, after which150 μL of wash buffer was added, and cells were pelleted at 1500 rpm for3 minutes and then resuspended in 100 μL of HBSS with 1% BSA. Sampleswere read on a FACS machine with a HTS attachment and the data wasanalyzed using Cell Quest Pro software. The results are summarized inTable 22, and data appears as Geometric Mean Shift values in arbitraryunits. These data demonstrate that at all concentrations tested, sc 264RAD had significantly less binding to parental 293T cells compared tothe original mAb sc 264.

TABLE 22 Cross-reactivity of mutated antibodies to parental cells.Concentration Antibody (ug/ml) 293T Cells 293T-αVβ6 Cells None n/a 3 2Mouse IgG2a 20 27 8 Human IgG1 20 4 4 Anti-aVb6 20 4 5 sc 264 20 4336673 sc 264 RAD 20 44 7241 sc 188 20 27 6167 sc 188 SDM 20 25 6758 sc264 5 88 6418 sc 264 RAD 5 13 6840 sc 188 5 9 5822 sc 188 SDM 5 9 6822sc 264 1.25 24 6230 sc 264 RAD 1.25 7 4890 sc 188 1.25 6 6395 sc 188 SDM1.25 5 4532

Example 22 Potency Analysis of Mutant Antibodies

In order to determine the concentration (IC₅₀) of mutant αVβ6 antibodiesrequired to inhibit TGFβLAP-mediated adhesion of HT-29 cells, thefollowing assay was performed.

Nunc MaxiSorp (Nunc) plates were coated overnight with 50 μL of 10 μg/mlTGF Beta1 LAP (TGFβLAP), and pre-blocked with 3% BSA/PBS for 1 hourprior to the assay. HT29 cells grown to the optimal density were thenpelleted and washed twice in HBBS (with 1% BSA and with 1 mM Ca²⁺ and 1mM Mg²⁺), after which the cells were then resuspended in HBSS at 30,000cell per well. The coating liquid was removed from the plates, whichwere then blocked with 100 μL 3% BSA at room temperature for 1 hour andthereafter washed twice with PBS.

Antibody titrations were prepared in 1:4 serial dilutions in a finalvolume of 30 μL and at two times the final concentration. Care was takento ensure that the PBS concentration in the control wells matched thePBS concentration in the most dilute antibody well. 30 μL of cells wereadded to each well, and the cells were incubated in the presence of theantibodies at 4° C. for 40 minutes in a V-bottom plate. Thecell-antibody mixtures were transferred to the coated plate and theplate was incubated at 37° C. for 40 minutes. The cells on the coatedplates were then washed four times in warm HBSS, and the cells werethereafter frozen at −80° C. for 15 minutes. The cells were allowed tothaw at room temperature, and then 100 μL of CyQuant dye/lysis buffer(Molecular Probes) was added to each well according to themanufacturer's instructions. Fluorescence was read at an excitationwavelength of 485 nm and an emission wavelength of 530 nm. The resultsfor twelve antibodies are summarized in Table 23, and demonstrate thatthe IC₅₀ of the mutant antibodies is consistently less than that of eachoriginal antibody.

TABLE 23 Concentration (IC₅₀) of mutant antibodies required to inhibitTGFβLAP-mediated adhesion of HT29 cells. n = 1 (ng/ml) n = 2 (ng/ml) n =3(ng/ml) sc.264 113 96 55 sc.264 RAD 13 13 39 sc.264 57 89 46 sc.188 125157 64 sc.188 SDM 22 24 67

Example 23 Cross-Reactivity of Mutant Antibodies to α4β1 Integrin

To establish that the mutant antibodies were functional only against theαVβ6 integrin and not the α4β1 integrin, an assay was performed to testthe ability of the antibodies to inhibit the adhesion of J6.77 cells tothe CS-1 peptide of fibronectin. The assay was performed as described asdescribed below.

Assay plates were coated with the CS-1 peptide of fibronectin. Assayplates were pre-blocked with 3% BSA/PBS for one hour prior to the assay.The J6.77 cells were grown to confluency, then removed from a cultureflask, pelleted and washed three times with HBSS. The cells were thenresuspended in HBSS at a concentration of 30,000 cells per well. Thecoating liquid containing the fibronectin fragments was removed, and theplates were blocked with 100 μL of 3% BSA for one hour at roomtemperature. The coated plates were washed three times with HBSS.Antibody titrations were prepared in 1:4 serial dilutions in a finalvolume of 30 μL and at twice the final concentration. The resuspendedcells were added to the wells containing the titrated antibody in aV-bottom plate, and the cells and antibodies were co-incubated at 4° C.for 40 minutes. The cell-antibody mixture was then transferred to thecoated plate, which was thereafter incubated at 37° C. for 40 minutes.The cells on the coated plates were next washed four times in warm HBSS,and the cells in the plates were then frozen at −80° C. for 15 minutes.The cells were allowed to thaw at room temperature, and then 100 μL ofCyQuant dye/lysis buffer (Molecular Probes) was added to each wellaccording to the manufacturer's instructions. Fluorescence was read atan excitation wavelength of 485 nm and an emission wavelength of 530 nm.

The results for the two mutant antibodies and their non-mutatedcounterparts are summarized in Table 24. A commercially available β1integrin specific antibody was used as a positive control in this assayand exhibited 95% inhibition of adhesion. A commercially available αVβ6specific antibody served as a negative control in this assay foradhesion to α4β1. All antibodies were used at 5 μg/ml and none of thetest antibodies could block adhesion to α4β1.

TABLE 24 Cross-Reactivity to α4β1 Integrin Antibody at 5ug/ml PercentInhibition sc.188 2 sc.188 SDM −6 sc.264 −30 sc.264 RAD −2 Human IgG1 26Human IgG2 13 Human IgG4 15 Anti-beta 1 Integrin 95

Example 24 Cross-Reactivity of Mutant Antibodies to α5β1 Integrin

To establish that the mutant antibodies were functional only against theαVβ6 integrin and not the α5β1 integrin, an assay was performed to testthe ability of the antibodies to inhibit the adhesion of K562 cells tofibronectin. The assay was performed as described as described inExample 14. The results are summarized in Table 25, and demonstrate thatnone of the tested antibodies could block adhesion to α5β1.

TABLE 25 Cross-Reactivity to α5β1 Integrin. Antibody ID % Inhibition sc188 −5 sc 188 SDM −8 sc 264 3 sc 264 RAD 6 αVβ6 Control −16 α5β1 Control78 Human IgG −12 Control

Example 25 Cross-Reactivity of Mutant Antibodies to Mouse and CynomolgusαVβ6 Integrin

In order to determine if the mutant αVβ6-specific antibodies exhibitcross-reactivity to mouse αVβ6 or Cynomolgus αVβ6, the following assaywas performed.

K562 parental cells, or K562 cells expressing Cynomolgus or mouse αVβ6were spun down after collection and resuspended in HBSS with 1% BSA and1 mM CaCl₂ and 1 mM MgCl₂ (wash buffer), so that at least 150,000 cellswere used in each reaction. Cells were divided between reactions in aV-bottom 96-well plate (Sarstedt), and the cells in the plate werepelleted at 1500 rpm for 3 minutes, after which the HBSS supernatant wasremoved. The primary antibody was added in a volume of 50 μL, and thecells were resuspended and thereafter incubated on ice for 60 minutes.After incubation, the cells were pelleted by centrifugation at 1500 rpmfor 3 minutes, resuspended in 100 μL wash buffer, and then pelletedagain. Cells were then resuspended in the appropriate secondary antibodyat 2 μg/ml with 10 μg/ml 7AAD, and stained on ice for 7 minutes, afterwhich 150 μL of wash buffer was added, and cells were pelleted at 1500rpm for 3 minutes and then resuspended in 100 μL of HBSS with 1% BSA.Samples were read on a FACS machine with a HTS attachment and the datawas analyzed using Cell Quest Pro software. The results are summarizedin Table 26, and data appears as Geometric Mean Shift values inarbitrary units. These data demonstrate that at the concentrationstested, sc 264 RAD and sc 188 SDM exhibit cross-reactivity to mouse andcynomolgus αVβ6.

TABLE 26 Cross-Reactivity with Mouse and Cynomolgus αVβ6 MouseCynomolgus Antibodies Parental alphaVbeta6 alphaVbeta6 Cells Alone 3 3 3Gt anti Mouse 5 6 7 anti 15 122 84 alphaVbeta6 anti alphaV 109 144 163anti beta6 26 43 37 Mouse IgG2a 23 36 25 Mouse IgG1 12 20 13 Gt antiHuman 7 12 7 Human IgG1 46 108 54 sc 133 57 246 154 sc 188 55 227 139 sc188 SDM 47 219 142 sc 254 98 260 190 sc 264 33 160 121 sc 264 RAD 48 196139 sc 298 33 150 97

Example 26 Internalization Assay

The internalization of the mutant antibodies was tested using a K562cell line that stably expressed human αVβ6. The assay was performed asdescribed in Example 15. Internalization of the purified antibodies wascompared to a commercially available αVβ6 antibody that was notinternalized in this assay.

The results are summarized in Table 27, and demonstrate that the sc 264RAD mutant antibody is internalized significantly less than thenon-mutated sc 264.

TABLE 27 Summary of the Internalization Assay Concentration PercentAntibody (ug/ml) Internalization sc 264 10 75% sc 264 1 47% sc 264 RAD10 42% sc 264 RAD 1 31% sc188 10 18% sc188 1 27% sc 188 SDM 10 22% sc188 SDM 1 17%

Example 27 Binding Affinity Analysis of SC 264 RAD Using FACS

The binding affinity to αVβ6 of the sc 264 RAD antibody was measured asdescribed in Example 18. The results of this assay are summarized inTable 28, and demonstrate that the sc 264 RAD antibody has an affinity<50 μM.

TABLE 28 Binding Affinity Analysis Using FACS mAb Sample K_(D) (pM) 95%CI (pM) sc 264 RAD 46.3 ±15.9

Example 28 Comparison of the Activity of SC 264 RAD with SC 264 RAD/ADY

The activity of sc 264 RAD antibody and the germlined (GL) version of264RAD (containing the mutation A84D in the light chain), 264 RAD/ADYwere compared in a Detroit-562 adhesion assay.

Plates were coated with 0.5 μg/ml GST-TGF-b LAP fusion protein at 4° C.overnight and the following morning, washed, and then blocked with 3%BSA/PBS for 1 hour. Detroit-562 cells (25000 cells per well) were thenallowed to adhere to the plates for 45 minutes at 37° C. in HBSScontaining 2 mM MgCl₂. After 45 minutes the plates were washed threetimes in PBS and then fixed in ethanol. Cells were visualized bystaining with Hoescht and quantitated by counting the number of cellsbound per well on a Cellomics Arrayscan II. The data shown in FIG. 5indicates that both sc 264 RAD and sc 264 RAD/ADY have similar activityand that the ability to block αVβ6 function is maintained in themodified antibody.

Example 29 Growth Study

To establish that the antibodies 264RAD, 133 and 188 SDM block avb6function in vivo each were tested for the ability to inhibit growth ofαV 6 positive tumour xenograft. One such model is the Detroit-562nasophayngeal cell line, which expresses αVβ6 and also grows as asub-cutaeneous tumour xenograft.

Detroit 562 cells were cultured in EMEM with Earle's BSS and 2 mML-Glu+1.0 mM sodium pyruvate, 0.1 mM NEAA+1.5 g/L sodium bicarbonate+10%FBS. Cells were harvested and resuspended in 50% PBS+50% matrigel. Thesuspension was then implanted at 5×10⁶ per mouse in a volume of 0.1 mlwithin the right flank. Animals were 6-8 week old NCR female nude mice.Dosing was initiated when tumours reached 0.1 cm3 and dosed at 20 mg/kgonce weekly for the duration of the study.

All three antibodies inhibited tumour growth (see FIG. 4). 264RAD wasthe most effective, followed by 133, and 188. This data clearly showsthat the antibodies 264RAD, 133 and 188 are active in vivo and are ablereduce the growth of a tumour dependent on αVβ6 signalling for growth.

INCORPORATION BY REFERENCE

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated herein byreference in their entirety for all purposes.

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The foregoingdescription and Examples detail certain preferred embodiments of theinvention and describes the best mode contemplated by the inventors. Itwill be appreciated, however, that no matter how detailed the foregoingmay appear in text, the invention may be practiced in many ways and theinvention should be construed in accordance with the appended claims andany equivalents thereof.

TABLE 29  Exemplary Antibody Heavy Chain Amino Acid Sequences SEQ ChainID Name NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 sc 264 75 QVQLQESGPGLVKGGSISSG WIRQHPG YIYYSGRTYNNP RVTISVDTSKNQFSLKLS VATGRADYH WGQGTTVT RADPSQTLSLTCTVS GYYWS KGLEWIG SLKS SVTAADTAVYYCAR FYAMDV VSS sc64 95QVQLQESGPGLVK GGSISSG WIRQHPG YIYYSGRTYNNP RVTISVDTSKNQFSLKLS VATGRADYHWGQGTTVT RAD/ADY PSQTLSLTCTVS GYYWS KGLEWIG SLKS SVTAADTAVYYCAR FYAMDVVSS sc 188 71 QVQLQESGPGLVK GGSISSG WIRQHPG YIYYSGSTSYNPRVTISVDTSKKQFSLKLT EGPLRGDYY WGQGTTVT SDM PSQTLSLTCTVS VYYWT NGLEWIGSLKS SVTAADTAVYYCAR YGLDV VSS sc 133 79 QVQLVQSGAEVKK GYTFTGY WVRQAPGWINPKSGDTNYA RVTMTRDTSTSTAYMELS RLDV WGQGTTVT TMT PGASVKVSCKAS YMHQGLEWMG QKFQG RLRSDDTAVYYCAR VSS sc 133 83 QVQLVQSGAEVKK GYTFTGY WVRQAPGWINPKSGDTNYA RVTLTRDTSTSTAYMELS RLDV WGQGTTVT WDS PGASVKVSCKAS YMHQGLEWMG QKFQG RLRSDDTAVYYCAR VSS sc 133 87 QVQLVQSGAEVKK GYTFTGY WVRQAPGWINPKSGDTNYA RVTMTRDTSTSTAYMELS RLDV WGQGTTVT TMT/WDS PGASVKVSCKAS YMHQGLEWMG QKFQG RLRSDDTAVYYCAR VSS

TABLE 30  Exemplary Antibody Light Chain Amino Acid Sequences SEQ ChainID Name NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 sc 264 77 SYELTQPSSVSVSSGDVLAK WFHQKPG KDSERPS GIPERFSGSSSGTTVTLT YSAADNNLV FGGGTKLT RADPGQTARITC KSAR QAPVLVIY ISGAQVEDEAAYYC VL sc 264 97 SYELTQPSSVSVSSGDVLAK WFHQKPG KDSERPS GIPERFSGSSSGTTVTLT YSAADNNLV FGGGTKLT RAD/ADYPGQTARITC KSAR QAPVLVIY ISGAQVEDEADYYC VL sc 188 73 EIVLTQSPGTLSLRAGQTIS WYQQKPG GASSRAT GIPDRFSGSGSGTDFTLT QQYGSSPRT FGQGTKVE SDMSPGERATLSC SRYLA QAPRPLIY ISRLEPEDFAVYYC IK sc 133 81 QSVLTQPPSVSAASGSSSNI WYQQLPG DNNKRPS GIPDRFSGSKSGTSATLG GTWNSSLSA FGTGTKVT TMTPGQKVTISC GNNYVS TAPKLLIY ITGLQTGDEADYYC GYV VL sc 133 85 QSVLTQPPSVSAASGSSSNI WYQQLPG DNNKRPS GIPDRFSGSKSGTSATLG GTWDSSLSA FGTGTKVT WDSPGQKVTISC GNNYVS TAPKLLIY ITGLQTGDEADYYC GYV VL sc 133 89 QSVLTQPPSVSAASGSSSNI WYQQLPG DNNKRPS GIPDRFSGSKSGTSATLG GTWDSSLSA FGTGTKVT TMT/WDSPGQKVTISC GNNYVS TAPKLLIY ITGLQTGDEADYYC GYV VL

What is claimed is:
 1. A method of treating cancer associated withincreased activation or expression of αVβ6 in an animal in need thereof,wherein the method comprises administering to the animal atherapeutically effective dose of an antibody that binds αVβ6, whereinthe antibody comprises a light chain variable region selected from thegroup consisting of: (a) a light chain sequence comprising the sequenceof SEQ ID NO:77; (b) a light chain sequence comprising the sequence ofSEQ ID NO:24; (c) a light chain sequence comprising the sequence of SEQID NO:40; and (d) a light chain sequence comprising the sequence of SEQID NO:28.
 2. The method according to claim 1, wherein the antibodycomprises the light chain sequence comprising SEQ ID NO:77.
 3. Themethod according to claim 1, wherein the antibody comprises the lightchain sequence comprising SEQ ID NO:24.
 4. A method of treating cancerassociated with increased activation or expression of αVβ6 in an animalin need thereof, wherein the method comprises administering to theanimal a therapeutically effective dose of an antibody that binds αVβ6,wherein the antibody comprises a heavy chain variable region selectedfrom the group consisting of: (a) a heavy chain sequence comprising thesequence of SEQ ID NO:75; (b) a heavy chain sequence comprising thesequence of SEQ ID NO:22; (c) a heavy chain sequence comprising thesequence of SEQ ID NO:38; and (d) a heavy chain sequence comprising thesequence of SEQ ID NO:26.
 5. The method according to claim 4, whereinthe antibody comprises the light chain sequence comprising SEQ ID NO:75.6. The method according to claim 4, wherein the antibody comprises thelight chain sequence comprising SEQ ID NO:22.
 7. A method of treatingcancer associated with increased activation or expression of αVβ6 in ananimal in need thereof, wherein the method comprises administering tothe animal a therapeutically effective dose of an antibody that bindsαVβ6, wherein the antibody comprises a heavy chain variable region and alight chain variable region selected from the group consisting of: (a) alight chain sequence comprising the sequence of SEQ ID NO:77 and a heavychain sequence comprising the sequence of SEQ ID NO:75; (b) a lightchain sequence comprising the sequence of SEQ ID NO:24 and a heavy chainsequence comprising the sequence of SEQ ID NO:22; (c) a light chainsequence comprising the sequence of SEQ ID NO:40 and a heavy chainsequence comprising the sequence of SEQ ID NO:38; and (d) a light chainsequence comprising the sequence of SEQ ID NO:28 and a heavy chainsequence comprising the sequence of SEQ ID NO:26.
 8. The methodaccording to claim 7, wherein the antibody comprises: (a) a heavy chainvariable region CDR1, CDR2, and CDR3 of SEQ ID NO:75; and (b) a lightchain variable region CDR1, CDR2 and CDR3 of SEQ ID NO:77.